Methods of treating inflammatory colon diseases

ABSTRACT

A method of treating ulcerative colitis or Crohn&#39;s disease in a subject in need thereof is disclosed. The method comprising administering to the subject a therapeutically effective amount of adherent cells from a placenta or adipose tissue, thereby treating the ulcerative colitis or Crohn&#39;s disease.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof treating inflammatory colon diseases using adherent cells fromadipose or placenta tissues and, more particularly, but not exclusively,to methods of treating ulcerative colitis or Crohn's disease using theadherent cells.

In the developing medical world a growing need exists for large amountsof adult stem cells for the purpose of cell engraftment and tissueengineering. In addition, adult stem cell therapy is continuouslydeveloping for treating and curing various conditions such ashematopoietic disorders, heart disease, Parkinson's disease, Alzheimer'sdisease, stroke, burns, muscular dystrophy, autoimmune disorders,diabetes and arthritis.

In recent years, considerable activity has focused on the therapeuticpotential of mesenchymal stromal cells (MSCs) for various medicalapplications including tissue repair of damaged organs such as thebrain, heart, bone and liver and in support of bone marrowtransplantations (BMT). MSCs, a heterogeneous population of cellsobtained from e.g. bone marrow, adipose tissue, placenta, and blood, iscapable of differentiating into different types of mesenchymal maturecells (e.g. reticular endothelial cells, fibroblasts, adipocytes,osteogenic precursor cells) depending upon influences from variousbioactive factors. Accordingly, MSCs have been widely studied inregenerative medicine as the foundation to build new tissues such asbone, cartilage and fat for the repair of injury or replacement ofpathologic tissues and as treatment for genetic and acquired diseases.Furthermore, the multipotent ability of MSCs, their easy isolation andculture, as well as their high ex vivo expansion potential make them anattractive therapeutic tool.

Inflammatory bowel disease (IBD), a group of inflammatory conditions ofthe large intestine and small intestine, includes Crohn's disease andulcerative colitis and is a chronic, relapsing, and remitting conditionof an unknown origin which affects at least 1 in 1,000 people in Westerncountries.

Crohn's disease (also known as granulomatous colitis and regionalenteritis), an autoimmune disease caused by the immune system'sattacking the gastrointestinal tract and producing inflammation in thegastrointestinal tract, is an inflammatory disease that may affect anypart of the gastrointestinal tract from mouth to anus, causing a widevariety of symptoms. It primarily causes abdominal pain, diarrhea,vomiting and weight loss, but may also cause complications outside ofthe gastrointestinal tract such as skin rashes, arthritis andinflammation of the eye. There is currently no known drug or surgicalcure for Crohn's disease and treatment options are restricted tocontrolling symptoms, maintaining remission and preventing relapse (e.g.5-aminosalicylic acid (5-ASA) formulations, corticosteroids such asprednisone and hydrocortisone, and immunomodulators such as azathioprineand mercaptopurine).

Ulcerative colitis, a form of colitis, is a disease of the intestine,specifically the large intestine or colon that includes characteristiculcers, or open sores, in the colon. The main symptom of active diseaseis usually constant diarrhea mixed with blood. Current treatment ofulcerative colitis includes anti-inflammatory drugs, immunosuppression,and biological therapy targeting specific components of the immuneresponse. Colectomy (partial or total removal of the large bowel throughsurgery) is occasionally necessary, and is considered to be a cure forthe disease.

Okamoto et al. [Okamoto et al., supra] and Matsumoto et al. [Matsumotoet al., Gastroenterology (2005) 128: 1851-1867] reported thatbone-marrow-derived cells (BMDCs) can repopulate the epithelia of thehuman gastrointestinal tract after graft-versus-host disease or gastriculcer formation following irradiation and bone marrow transplantation.Komori et al. 2005 [Komori et al., J Gastroenterol (2005) 40: 591-599]also reported transient increases in bone-marrow-derived mucosalepithelial cells and myofibroblasts during the healing process ofgastric ulcers and trinitrobenzene sulfonic acid (TNBS)-induced colitisin rats. In addition, Osiris therapeutics (www.osiris.com) is evaluatingProchymal, a product derived from bone marrow MSCs, for the treatment ofCrohn's disease. Osiris is currently conducting a multi-center trial toevaluate the safety and efficacy of Prochymal for Crohn's disease.

PCT Publication No. WO 2008/100498 discloses methods of treatingimmune-related diseases (e.g. inflammatory bowel disease,graft-versus-host disease) using placental stem cells or umbilical cordstem cells. The stem cells disclosed are derived from a mammalianplacenta, regardless of morphology, cell surface markers or the numberof passages after a primary culture, and adhere to a tissue culturesubstrate (e.g., tissue culture plastic or a fibronectin-coated tissueculture plate).

U.S. Publication No. 20080213227 discloses methods of treatingautoimmune diseases and inflammatory diseases (e.g. inflammatory boweldisease and Crohn's disease) by administering mesenchymal stem cells inan effective amount. The mesenchymal cells disclosed may be obtainedfrom adherent marrow or periosteal cells or alternatively from blood,skin, cord blood, muscle, fat, bone, or perichondrium.

PCT Publication No. WO 2007/108003 discloses methods of cell expansion,which comprise culturing adherent cells from placenta or adipose tissueunder three-dimensional culturing conditions, which support cellexpansion. Also provided are cells generated thereby and uses of same.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of treating ulcerative colitis or Crohn'sdisease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount ofadherent cells from a placenta or adipose tissue, thereby treating theulcerative colitis or Crohn's disease.

According to an aspect of some embodiments of the present inventionthere is provided a use of adherent cells from a placenta or adiposetissue for the manufacture of a medicament identified for treatingulcerative colitis or Crohn's disease.

According to an aspect of some embodiments of the present inventionthere is provided an article of manufacture comprising a packagingmaterial which comprises a label for use in treating ulcerative colitisor Crohn's disease, the packaging material packaging a pharmaceuticallyeffective amount of adherent cells from a placenta or adipose tissue.

According to some embodiments of the invention, the adherent cellscomprise a positive marker expression selected from the group consistingof CD73, CD90, CD29 and CD105.

According to some embodiments of the invention, the adherent cellscomprise a negative marker expression selected from the group consistingof CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 and CD79.

According to some embodiments of the invention, the adherent cells arecapable of suppressing an immune reaction.

According to some embodiments of the invention, suppressing an immunereaction comprises suppressing a T cell activity.

According to some embodiments of the invention, the adherent cells areobtained from a three-dimensional (3D) culture.

According to some embodiments of the invention, the three-dimensional(3D) culture comprises a 3D bioreactor.

According to some embodiments of the invention, culturing of theadherent cells in the 3D culture is effected under perfusion.

According to some embodiments of the invention, culturing of theadherent cells is effected for at least 3 days.

According to some embodiments of the invention, culturing of theadherent cells is effected until at least 10% of the adherent cells areproliferating.

According to some embodiments of the invention, the adherent cellscomprise a gene expression profile as described in Table 11.

According to some embodiments of the invention, the adherent cellscomprise cells cultured from the placenta or adipose tissue under 2dimensional (2D) culturing conditions.

According to some embodiments of the invention, at least 12% of theadherent cells are at a S and/or G2/M proliferative phase.

According to some embodiments of the invention, the adherent cellscomprise a gene expression profile as described in Table 8.

According to some embodiments of the invention, the adherent cells areless committed to an osteogenic lineage as compared to adherent cellsfrom bone marrow grown and allowed to differentiate under the sameconditions.

According to some embodiments of the invention, the adherent cells areless committed to an adipogenic lineage as compared to adherent cellsfrom bone marrow grown and allowed to differentiate under the sameconditions.

According to some embodiments of the invention, the article ofmanufacture further comprises an additional drug for treatment of coloninflammation.

According to some embodiments of the invention, the article ofmanufacture further comprises an immunosuppressant agent.

According to some embodiments of the invention, the article ofmanufacture further comprises an anti-inflammatory agent.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B are figures depicting cell cycle analysis of 2D adherentcells of placenta suitable for use in accordance with the presentteachings (FIG. 1A) or adherent cells manufactured according to theteachings of WO/2007/108003, designated PDC (FIG. 1B). Cells were fixedin 70% EtOH O.N, centrifuged and re-suspended in a Propidium Iodide (PI)solution and then analyzed by FACS.

FIG. 2 is a bar graph depicting marker expression on 2D adherent cellsof placenta suitable for use in accordance with the present teachings.Of note, negative expression was recorded for CD11b, CD34, HLA-DR, CD14,CD19 and CD45, while positive expression was noted for CD29, CD73, CD90and CD105.

FIG. 3 is a bar graph depicting reduction of lymphocyte cell response by2D adherent cells of placenta suitable for use in accordance with thepresent teachings. Peripheral blood (PB) derived mononuclear cells(MNCs) were stimulated with PHA (10 μg/ml). One of four differentbatches of 2D adherent cells were added to the stimulated MNCs. Threereplicates of each group were seeded in 96-well plates.

FIGS. 4A-F are photographs depicting growth of bone marrow and placentacells under osteogenesis or adipogenesis differentiation conditions.Bone marrow derived cells (FIGS. 4A-C) or placenta derived cells (FIGS.4D-F) were plated in growth medium (FIGS. 4A and 4D), osteogenesisdifferentiation medium (FIGS. 4B and 4E) or adipogenesis differentiationmedium (FIGS. 4C and 4F) in a 24 well plate coated with vitronectin andcollagen. Medium was replaced every 3-4 days. At the end of growthperiod cells were fixed, stained and pictured as described in detail theExamples section which follows.

FIGS. 5A-F are photographs depicting growth of bone marrow and placentacells under modified osteogenesis or adipogenesis differentiationconditions. Bone marrow derived cells (FIGS. 5A-C) or placenta derivedcells (FIGS. 5D-F) were plated in growth medium (FIGS. 5A and 5D),osteogenesis differentiation medium (FIGS. 5B and 5E) or adipogenesisdifferentiation medium (FIGS. 5C and 5F) in a 24 well plate coated withvitronectin and collagen. Medium was replaced every 3-4 days. At the endof growth period cells were fixed, stained and pictured as described indetail the Examples section which follows.

FIGS. 6A-B depict cell cycle analysis of 3D adherent cells manufactureby Plurix (designated PLX, FIG. 6B) and by Celligen (PLX-C, FIG. 6A).Cells were fixed in 70% EtOH O.N, centrifuged and re-suspended in aPropidium Iodide (PI) solution and then analyzed by FACS.

FIGS. 7A-C depict expression of fibroblast-typical markers but notexpression of endothelial typical markers on PLX-C. FIG. 7A depictsnegative expression of the endothelial marker CD31; FIG. 7B depictsnegative expression of the endothelial marker KDR; and FIG. 7C depictspositive expression of the human fibroblast marker (D7-FIB). Of note,the red histograms for Isotype IgG1 (FITC) represent the negativecontrol while the blue histograms represent the positively stainedcells.

FIGS. 8A-D depict expression of stimulatory and co-stimulatory moleculeson PLX-C cells. FIG. 8A depicts PLX-C expression of CD80; FIG. 8Bdepicts PLX-C expression of CD86; FIG. 8C depicts PLX-C expression ofCD40; and FIG. 8D depicts PLX-C expression of HLA-AB/C. Negativecontrols were prepared with relevant isotype fluorescence molecules. Ofnote, red histograms indicate PLX-C marker-expressing population ofcells, blue histograms indicate bone marrow (BM) marker-expressingpopulation of cells, and green histograms indicate mononuclear cell(MNC) marker expressing population of cells.

FIGS. 9A-B depict inhibition of lymphocyte proliferation by PLX-C. FIG.9A depicts Mixed Lymphocyte Reaction (MLR) tests performed with 2×10⁵peripheral blood (PB) derived mononuclear cells (MNC, donor A)stimulated with equal amount of irradiated (3000 Rad) PB derived MNCs(donor B) followed by addition of increasing amounts of PLX-C cells tothe cultures. Three replicates of each group were seeded in 96-wellplates. Proliferation rate was measured by [³H]thymidine incorporation;FIG. 9B depict peripheral blood (PB) derived MNCs stimulated with ConA(1.5 mg/ml). Increasing amounts of PLX-C cells were added to thecultures. Three replicates of each group were seeded in 96-well plates.Proliferation rate was measured by [³H]thymidine incorporation.

FIGS. 10A-C depict PLX-C regulation of pro-inflammatory andanti-inflammatory cytokine secretion following co-culture withperipheral blood cells. FIGS. 10A-B depict secretion of IFNγ (FIG. 10A)and TNFα (FIG. 10B) following co-culture of human derived MNCs (isolatedfrom peripheral blood) stimulated with ConA with PLX-C; FIG. 10C depictssecretion of IFNγ, TNFα and IL-10 following co-culture of human derivedMNCs (isolated from peripheral blood) stimulated with LPS with PLX-C.Supernatants were collected and subjected to cytokines analysis usingELISA.

FIG. 11 is a graph depicting macroscopic evaluation of colon tissues ofcolitic mice as represented by the Wallace score. TNBS (colitis modelmice), TNBS+5-ASA (colitic mice who received the gold standardtreatment), TNBS+2D adherent cells (batch 1) ip, TNBS+3D adherent cells(PLX-C, batch 2) ip, TNBS+2D adherent cells (batch 1) iv and TNBS+3Dadherent cells (PLX-C, batch 2) iv. Macroscopic assessments wereconducted blindly by two investigators.

FIG. 12 is a graph depicting microscopic evaluation of colon tissues ofcolitic mice as represented by the Ameho score. TNBS (colitis modelmice), TNBS+5-ASA (colitic mice who received the gold standardtreatment), TNBS+2D adherent cells (batch 1) ip, TNBS+3D adherent cells(PLX-C, batch 2) ip, TNBS+2D adherent cells (batch 1) iv and TNBS+3Dadherent cells (PLX-C, batch 2) iv. Histological assessments wereconducted blindly by two investigators.

FIG. 13 is a graph depicting IL-1β mRNA expression level in colonictissues of colitic mice. Mice were rendered colitic by intrarectaladministration of TNBS and were administered 2D or 3D (PLX-C) adherentcells via intraperitoneal or intravenous routes. Total RNA was isolatedfrom colonic tissues of the different experimental groups and IL-1βexpression levels were evaluated by RT-PCR.

FIG. 14 is a graph depicting microscopic evaluation of colon tissues ofcolitic rats. Rats were rendered colitic by intracolonic administrationof TNBS and were administered PLX-C cells via intraperitoneal (ip) orintravenous (iv) routes.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to methodsof treating inflammatory colon diseases using adherent cells fromadipose or placenta tissues and, more particularly, but not exclusively,to methods of treating ulcerative colitis or Crohn's disease using theadherent cells.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

While reducing the present invention to practice, the present inventorhas surprisingly uncovered that adherent cells from placental tissuesmay be used effectively to treat uleceritive colitis and Crohn'sdisease.

As is shown hereinbelow and in the Examples section which follows, thepresent inventor has uncovered through laborious experimentation thatadherent cells obtained from placenta or adipose tissues and culturedunder 2D (Example 2) or 3D culturing conditions (Examples 1 and 3) maybe effectively used to treat colon inflammation, such as ulcerativecolitis, as depicted in both mouse (Example 4) and rat (Example 5)experimental models. The present inventor has shown that intravenous(iv) or intraperitoneal (ip) administration of the 2D or 3D adherentcells of the present invention resulted in a major improvement in theinflammatory condition of the colon tissue as determined by bothmacroscopic and microscopic evaluations of the colon (FIGS. 11, 12 and14). This anti-inflammatory effect was as efficient as the 5-ASA goldstandard treatment. Taken together the present teachings portray ananti-inflammatory value for the adherent cells of the present inventionand suggest the use of same for the treatment of inflammatory colondiseases such as ulcerative colitis and Crohn's disease.

Thus, according to one aspect of the present invention there is provideda method of treating ulcerative colitis or Crohn's disease in a subjectin need thereof, the method comprising administering to the subject atherapeutically effective amount of adherent cells from a placenta oradipose tissue, thereby treating the ulcerative colitis or Crohn'sdisease.

As used herein the term “treating” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of ulcerative colitis or Crohn's disease. Thoseof skill in the art will understand that various methodologies andassays can be used to assess the development of a pathology, andsimilarly, various methodologies and assays may be used to assess thereduction, remission or regression of a pathology.

The term “ulcerative colitis” as used herein refers to a medicalcondition of the intestine, a form of inflammatory bowel disease (IBD),specifically of the large intestine or colon, that includescharacteristic ulcers, or open sores, in the colon. Ulcerative colitisdisease is usually diagnosed following recurrent symptoms of constantdiarrhea mixed with blood, of gradual onset. Ulcerative colitisaccording to the present teachings refers to any stage or severity ofulcerative colitis (e.g. disease remission or acute disease).

The term “Crohn's disease” as used herein refers to an inflammatorycondition that may affect any part of the gastrointestinal tract frommouth to anus, also known as granulomatous colitis or regionalenteritis, and is a form of inflammatory bowel disease (IBD). Crohn'sdisease is a type of autoimmune disease and is usually diagnosedfollowing recurrent symptoms of abdominal pain, diarrhea (which may bebloody), vomiting, weight loss, skin rashes, arthritis and inflammationof the eye. Crohn's disease according to the present teachings refers toany stage or severity of Crohn's disease (e.g. disease remission, acutedisease, relapse).

As used herein the phrase “a subject in need thereof” refers to amammal, preferably a human subject, male or female of any age, who hasbeen diagnosed with probable or definite ulcerative colitis or Crohn'sdisease, e.g., a subject who experienced inflammatory colon disease. Thediagnosis of ulcerative colitis or Crohn's disease may include anydiagnosis test as, for example, laboratory tests, endoscopic evaluation,biopsies of the mucosa (for ulcerative colitis), barium follow-throughx-ray (for Crohn's disease), and CT or MRI scans (for Crohn's disease).

It will be appreciated that the present invention also envisiontreatment of other colon inflammatory conditions, including, but are notlimited to, chronic inflammatory intestinal diseases (Garcia Herola A.et al., Gastroenterol. Hepatol. 2000 January; 23 (1):16), celiac disease(Landau Y E. and Shoenfeld Y. Harefuah 2000 Jan. 16; 138 (2):122) andileitis using the adherent cells of the present invention.

As mentioned hereinabove, the method, according to this aspect of thepresent invention, is effected by administering to the subject atherapeutically effective amount of adherent cells from a placenta oradipose tissue.

As used herein the phrase “adherent cells” refers to a homogeneous orheterogeneous population of cells which are anchorage dependent, i.e.,require attachment to a surface in order to grow in vitro.

As used herein the phrase “adipose tissue” refers to a connective tissuewhich comprises fat cells (adipocytes).

As used herein the term “placenta tissue” refers to any portion of themammalian female organ which lines the uterine wall and during pregnancyenvelopes the fetus, to which it is attached by the umbilical cord.Following birth, the placenta is expelled (and is referred to as a postpartum placenta). In an exemplary embodiment, placenta refers to wholeplacenta.

According to the present teachings, placenta or adipose tissue derivedadherent cells can be propagated using two dimensional (2D) or threedimensional (3D) culturing conditions.

As used herein the phrase “two dimensional culture” refers to a culturein which the cells are disposed to conditions which are compatible withcell growth while allowing the cells to grow in one plane. Theconditions in the two dimensional culture of the invention are designedto enable expansion of the adherent cells.

As used herein the phrase “three dimensional culture” refers to aculture in which the cells are disposed to conditions which arecompatible with cell growth while allowing the cells to grow in morethan one layer. It is well appreciated that the in situ environment of acell in a living organism (or a tissue) is in a three dimensionalarchitecture. Cells are surrounded by other cells. They are held in acomplex network of extra cellular matrix nanoscale fibers that allowsthe establishment of various local microenvironments. Their extracellular ligands mediate not only the attachment to the basal membranebut also access to a variety of vascular and lymphatic vessels. Oxygen,hormones and nutrients are ferried to cells and waste products arecarried away. The conditions in the three dimensional culture of theinvention are designed to mimic such an environment as is furtherexemplified below.

It will be appreciated that the conditions of the two-dimensional andthree-dimensional cultures are such that enable expansion of theadherent cells.

As used herein the terms “expanding” and “expansion” refer tosubstantially differentiation-less maintenance of the cells andultimately cell growth, i.e., increase of a cell population (e.g., atleast 2 fold) without differentiation accompanying such increase.

As used herein the terms “maintaining” and “maintenance” refer tosubstantially differentiation-less cell renewal, i.e., substantiallystationary cell population without differentiation accompanying suchstationarity.

As mentioned, the adherent cells of this aspect of the invention areretrieved from an adipose or placental tissue.

Placental cells may be obtained from a full-term or pre-term placenta.Placenta is preferably collected once it has been ex blooded. Theplacenta is preferably perfused for a period of time sufficient toremove residual cells. The term “perfuse” or “perfusion” used hereinrefers to the act of pouring or passaging a fluid over or through anorgan or tissue. The placental tissue may be from any mammal; forexample, the placental tissue is human. A convenient source of placentaltissue is from a post partum placenta (e.g., 1-6 hours), however, thesource of placental tissue or cells or the method of isolation ofplacental tissue is not critical to the invention.

Placenta derived adherent cells may be obtained from both fetal (i.e.,amnion or inner parts of the placenta, see Example 1) and maternal(i.e., decidua basalis, and decidua parietalis) parts of the placenta.Tissue specimens are washed in a physiological buffer [e.g.,phosphate-buffered saline (PBS) or Hank's buffer]. Single-cellsuspensions are made by treating the tissue with a digestive enzyme (seebelow) or/and mincing and flushing the tissue parts through a nylonfilter or by gentle pipetting (Falcon, Becton, Dickinson, San Jose,Calif.) with washing medium.

Adipose tissue derived adherent cells may be isolated by a variety ofmethods known to those skilled in the art. For example, such methods aredescribed in U.S. Pat. No. 6,153,432. The adipose tissue may be derivedfrom omental/visceral, mammary, gonadal, or other adipose tissue sites.One source of adipose tissue is omental adipose. In humans, the adiposeis typically isolated by liposuction.

Isolated adherent cells from placenta or adipose tissue may be derivedby treating the tissue with a digestive enzyme such as collagenase,trypsin and/or dispase; and/or effective concentrations of hyaluronidaseor DNAse; and ethylenediaminetetra-acetic acid (EDTA); at temperaturesbetween 25-50° C., for periods of between 10 minutes to 3 hours. Thecells may then be passed through a nylon or cheesecloth mesh filter ofbetween 20 microns to 1 mm. The cells are then subjected to differentialcentrifugation directly in media or over a Ficoll or Percoll or otherparticulate gradient. Cells are centrifuged at speeds of between 100 to3000×g for periods of between 1 minutes to 1 hour at temperatures ofbetween 4-50° C. (see U.S. Pat. No. 7,078,230).

In addition to placenta or adipose tissue derived adherent cells, theinvention also envisages the use of adherent cells from other cellsources which are characterized by stromal stem cell phenotype (as willbe further described herein below). Tissue sources from which adherentcells can be retrieved include, but are not limited to, cord blood,scalp, hair follicles [e.g. as described in Us Pat. App. 20060172304],testicles [e.g., as described in Guan K., et al., Nature. 2006 Apr. 27;440(7088):1199-203], human olfactory mucosa [e.g., as described inMarshall, C T., et al., Histol Histopathol. 2006 June; 21(6):633-43],embryonic yolk sac [e.g., as described in Geijsen N, Nature. 2004 Jan.8; 427(6970):148-54] and amniotic fluid [Pieternella et al. (2004) StemCells 22:1338-1345], all of which are known to include mesenchymal stemcells. Adherent cells from these tissue sources can be isolated byculturing the cells on an adherent surface, thus isolating adherentcells from other cells in the initial population.

Regardless of the origin (e.g., placenta or adipose tissue), cellretrieval is preferably effected under sterile conditions. Once isolatedcells are obtained, they are allowed to adhere to an adherent material(e.g., configured as a surface) to thereby isolate adherent cells.Culturing then proceeds under 2D conditions (as described in Example 2of the Examples section) and cells may be further transferred to 3Dconditions (as described in Examples 1 and 3 of the Examples section).

As used herein “an adherent material” refers to a synthetic, naturallyoccurring or a combination of same of a non-cytotoxic (i.e.,biologically compatible) material having a chemical structure (e.g.,charged surface exposed groups) which may retain the cells on a surface.

Examples of adherent materials which may be used in accordance with thisaspect of the invention include, but are not limited to, a polyester, apolypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinylchloride, a polystyrene, a polysulfone, a cellulose acetate, a glassfiber, a ceramic particle, a matrigel, an extra cellular matrixcomponent (e.g., fibronectin, chondronectin, laminin), a collagen, apoly L lactic acid and an inert metal fiber.

It will be appreciated that seeding of placenta or adipose cells istypically effected at a culture density of 3±0.2×10³ cells/cm².Following seeding, cell cultures are usually cultured in a tissueculture incubator under humidified conditions with 5% CO2 at 37° C.

Further steps of purification or enrichment for stromal stem cells maybe effected using methods which are well known in the art (such as byFACS using stromal stem cell marker expression, as further describedherein below).

Non-limiting examples of base media useful in culturing according to theinvention include Minimum Essential Medium Eagle, ADC-1, LPM (BovineSerum Albumin-free), F10(HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJMedium (with and without Fitton-Jackson Modification), Basal MediumEagle (BME—with the addition of Earle's salt base), Dulbecco's ModifiedEagle Medium (DMEM—without serum), Yamane, IMEM-20, Glasgow ModificationEagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, MediumM199 (M199E—with Earle's sale base), Medium M199 (M199H—with Hank's saltbase), Minimum Essential Medium Eagle (MEM-E—with Earle's salt base),Minimum Essential Medium Eagle (MEM-H—with Hank's salt base) and MinimumEssential Medium Eagle (MEM-NAA with non essential amino acids), amongnumerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066,NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell,Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153. Apreferred medium for use in the invention is DMEM. These and otheruseful media are available from GIBCO, Grand Island, N.Y., USA andBiological Industries, Bet HaEmek, Israel, among others. A number ofthese media are summarized in Methods in Enzymology, Volume LVIII, “CellCulture”, pp. 62 72, edited by William B. Jakoby and Ira H. Pastan,published by Academic Press, Inc.

The medium may be supplemented such as with serum such as fetal serum ofbovine or other species, and optionally or alternatively, growthfactors, vitamins (e.g. ascorbic acid), cytokines, salts (e.g.B-glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g.,growth hormone, erythropoeitin, thrombopoietin, interleukin 3,interleukin 6, interleukin 7, macrophage colony stimulating factor,c-kit ligand/stem cell factor, osteoprotegerin ligand, insulin, insulinlike growth factors, epidermal growth factor, fibroblast growth factor,nerve growth factor, cilary neurotrophic factor, platelet derived growthfactor, and bone morphogenetic protein at concentrations of betweenpicogram/ml to milligram/ml levels.

It is further recognized that additional components may be added to theculture medium. Such components may be antibiotics, antimycotics,albumin, amino acids, and other components known to the art for theculture of cells. Additionally, components may be added to enhance thedifferentiation process when needed (see further below).

It will be appreciated that in case the adherent cells of the inventionare administered to a human subject, the cells and the culture medium(e.g., with the above described medium additives) should besubstantially xeno-free, i.e., devoid of any animal contaminants e.g.,mycoplasma. For example, the culture medium can be supplemented with aserum-replacement, human serum and/or synthetic or recombinantlyproduced factors.

As mentioned, once adherent cells are at hand they may be passaged to 2Dor 3D settings (see Examples 1, 2 and 3 of the Examples section whichfollows). It will be appreciated though, that the cells may betransferred to a 3D-configured matrix immediately after isolation oralternatively, may be passaged to 3D settings following 2D conditions(as mentioned hereinabove).

It will be appreciated that during the 2D culturing conditions, theadherent cells may be continuously passaged. According to an embodimentof the present invention, the cells may be passaged for at least 4passages, at least 5 passages, at least 6 passages, at least 7 passagesor at least 8 passages. It will be appreciated that cells are typicallypassaged when the culture reaches about 70-80% confluence, typicallyafter 3-5 days (1.5-2 doublings). Moreover, under the 2D culturingconditions, the cells may be grown in a culture medium devoid ofantibiotic supplements from at least passage 2, at least passage 3, orat least passage 4.

Thus, during the 2D culture, culturing is effected for at least about 2days, 3 days, 4 days, 5 days, 10 days, 20 days, a month or even more.Passaging may also be effected to increase cell number. It will beappreciated that culture medium may be changed in order to prolong andimprove culturing conditions.

The 2D adherent cells can be harvested when at least about 12% of cellsare proliferating while avoiding uncontrolled differentiation andsenescence.

2D adherent cells of some embodiments of the present invention compriseat least about 10%, 28%, 30%, 50%, 80% or more proliferative cells (ascan be assayed by FACS monitoring S and/or G2/M phases).

As mentioned, the adherent cells may be transferred to 3D settings.

Thus, the adherent material of this aspect of the invention isconfigured for 3D culturing thereby providing a growth matrix thatsubstantially increases the available attachment surface for theadherence of the cells so as to mimic the infrastructure of the tissue(e.g., placenta).

For high scale production, culturing can be effected in a 3D bioreactor.

Examples of such bioreactors include, but are not limited to, a plugflow bioreactor, a continuous stirred tank bioreactor, a stationary-bedbioreactor, a CelliGen Plus® bioreactor system (New Brunswick Scientific(NBS) or a BIOFLO 310 bioreactor system (New Brunswick Scientific (NBS).

As shown Example 3 of the Examples section, the Celligen bioreactor iscapable of 3D expansion of adherent cells under controlled conditions(e.g. pH, temperature and oxygen levels) and with constant cell growthmedium perfusion. Furthermore, the cell cultures can be directlymonitored for concentration levels of glucose, lactate, glutamine,glutamate and ammonium. The glucose consumption rate and the lactateformation rate of the adherent cells enable to measure cell growth rateand to determine the harvest time.

Other 3D bioreactors that can be used with the invention include, butare not limited to, a continuous stirred tank bioreactor, where aculture medium is continuously fed into the bioreactor and a product iscontinuously drawn out, to maintain a time-constant steady state withinthe reactor. A stirred tank bioreactor with a fibrous bed basket isavailable for example at New Brunswick Scientific Co., Edison, N.J.), Astationary-bed bioreactor, an air-lift bioreactor, where air istypically fed into the bottom of a central draught tube flowing up whileforming bubbles, and disengaging exhaust gas at the top of the column],a cell seeding perfusion bioreactor with Polyactive foams [as describedin Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)] tubularpoly-L-lactic acid (PLLA) porous scaffolds in a Radial-flow perfusionbioreactor [as described in Kitagawa et al., Biotechnology andBioengineering 93(5): 947-954 (2006). Other bioreactors which can beused in accordance with the invention are described in U.S. Pat. Nos.6,277,151, 6,197,575, 6,139,578, 6,132,463, 5,902,741 and 5,629,186.

Cell seeding is preferably effected 100,000-1,500,000 cells/mm atseeding. In an exemplary embodiment a total of 150±30×10⁶ cells areseeded, 3-5×10⁶ cell/gr carrier are seeded, or 0.015-0.1×10⁶ cell/ml areseeded.

Culturing is effected for at least about 2 days, 3 days, 4 days, 5 days,10 days, 20 days, a month or even more. It will be appreciated thatculturing in a bioreactor may prolong this period. Culturing of theadherent cells in the 3D culture can be effected under a continuous flowof a culture medium. Passaging may also be effected to increase cellnumber. It will be appreciated that culture medium may be changed inorder to prolong and improve culturing conditions.

According to an embodiment of the present invention, culturing theadherent cells in a 3D culture may be effected under perfusion of theculture medium. Typically, the perfusion rate is determined by theglucose concentration in the culture medium of the adherent cells. Thus,according to the present teachings, the culture medium may be changedwhen the glucose concentration is about 500 mg/L, about 550 mg/L, orabout 600 mg/L.

The 3D adherent cells can be harvested when at least about 10% of cellsare proliferating while avoiding uncontrolled differentiation andsenescence. 3D adherent cells of some embodiments of the presentinvention comprise at least about 10%, 28%, 30%, 50%, 80% or moreproliferative cells (as can be assayed by FACS monitoring S and/or G2/Mphases).

Adherent cells of some embodiments of the invention may comprise atleast one “stromal stem cell phenotype”.

As used herein “a stromal stem cell phenotype” refers to a structural orfunctional phenotype typical of a bone-marrow derived stromal (i.e.,mesenchymal) stem cell

As used herein the phrase “stem cell” refers to a cell which is notterminally differentiated.

Thus for example, the cells may have a spindle shape. Alternatively oradditionally the cells may express a marker or a collection of markers(e.g. surface marker) typical to stromal stem cells. Examples of stromalstem cell surface markers (positive and negative) include but are notlimited to CD105+, CD29+, CD44+, CD73+, CD90+, CD3−, CD4−, CD34−, CD45−,CD80−, CD19−, CD5−, CD20−, CD11B−, CD14−, CD19−, CD79−, HLA-DR−, andFMC7−. Other stromal stem cell markers include but are not limited totyrosine hydroxylase, nestin and H—NF.

According to a specific embodiment of the present invention, theadherent cells do not express Oct-4.

It will be appreciated that the 2D adherent cells of placenta tissuegenerated according to the present teachings have a gene expressionprofile essentially as described in Table 8 of the Examples sectionwhich follows. While, the 3D adherent cells of placenta tissue generatedaccording to the present teachings have a gene expression profileessentially as described in Table 11 of the Examples section whichfollows.

According to an exemplary embodiment, the 2D and 3D adherent cells ofthe present invention are less committed to differentiation intoosteogenic or adipogenic lineages as compared to adherent cells from thebone marrow grown and differentiated under the same conditions.

Examples of functional phenotypes typical of stromal stem cells include,but are not limited to, T cell suppression activity (they don'tstimulate T cells and conversely suppress same) and hematopoietic stemcell support activity.

According to one embodiment of the invention, the adherent cells of theinvention are capable of suppressing immune reaction in a subject.

As used herein the phrase “suppressing immune reaction in a subject”refers to decreasing or inhibiting the immune reaction occurring in asubject in response to an antigen (e.g., a foreign cell or a portionthereof). The immune response which can be suppressed by the adherentcells include the humoral immune responses, and cellular immuneresponses, which involve specific recognition of pathogen antigens viaantibodies and T-lymphocytes (proliferation of T cells), respectively.

As is shown in Examples 4-5 of the Examples section which follows, the2D and 3D adherent cells of the present invention were found to inducean anti-inflammatory effect in colon inflammatory conditions. It will befurther appreciated that this effect may be mediated by the cells per seor by a factor secreted thereby having an anti-inflammatory effect evenin the absence of cells. Thus, the adherent cells of the presentinvention may be preferentially used in treating intestinalinflammation, such as in conditions of ulcerative colitis and Crohn'sdisease.

The phrase “administering to the subject” refers to the introduction ofthe cells of the invention to target tissue. The cells can be derivedfrom the recipient or from an allogeneic or xenogeneic donor. Thisphrase also encompasses “transplantation”, “cell replacement” or“grafting” of the cells of the invention into the subject.

According to specific embodiments of the invention, the adherent cellsmay be administered to the subject by any means known to one of ordinaryskill in the art, for example, by intravenous (iv), intramuscular (im),or intraperitoneal (ip) administration.

Cells which may be administered in accordance with this aspect of theinvention include the above-described adherent cells which may becultured in three-dimensional or two dimensional settings as well asmesenchymal and-non mesenchymal partially or terminally differentiatedderivatives of same.

Methods of deriving lineage specific cells from the stromal stem cellsof the invention are well known in the art. See for example, U.S. Pat.Nos. 5,486,359, 5,942,225, 5,736,396, 5,908,784 and 5,902,741.

The cells may be naïve or genetically modified such as to derive alineage of interest (see U.S. Pat. Appl. No. 20030219423).

The cells may be of autologous or non-autologous source (i.e.,allogeneic or xenogeneic) of fresh or frozen (e.g., cryo-preserved)preparations.

Since non-autologous cells may induce an immune reaction whenadministered to the body several approaches have been developed toreduce the likelihood of rejection of non-autologous cells. Theseinclude either suppressing the recipient immune system or encapsulatingthe non-autologous cells in immunoisolating, semipermeable membranesbefore transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesTechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al., Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).Examples of immunosuppressive agents which may be used include, but arenot limited to, methotrexate, cyclophosphamide, cyclosporine,cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine(sulphasalazopyrine), gold salts, D-penicillamine, leflunomide,azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha.blockers, a biological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

Depending on the medical condition, the subject may be administered withadditional chemical drugs (e.g., immunomodulatory, chemotherapy,anti-inflammatory etc.) or cells.

For the treatment of inflammatory colon conditions, including ulcerativecolitis and Crohn's disease, any treatment known to one of ordinaryskill in the art may be employed, including for example,Aminosalicylates (e.g. Sulfasalazine, Mesalazine, Balsalazide,Olsalazine), Corticosteroids (e.g. Cortisone, Prednisone, Prednisolone,Cortifoam, Hydrocortisone, Methylprednisolone, Beclometasone,Budesonide), Immunosuppressive drugs (e.g. Mercaptopurine, Azathioprine,Methotrexate, Tacrolimus), Biological treatments (e.g. Infliximab,Visilizumab), Low Molecular Weight Heparin (LMWH), dietary modifications(e.g. fibers) and surgery.

The subject may also be administered an anti-inflammatory agent such as,but not limited to, Alclofenac; Alclometasone Dipropionate; AlgestoneAcetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium;Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone;Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride;Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone;Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac;Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort;Desonide; Desoximetasone; Dexamethasone Dipropionate; DiclofenacPotassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac;Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap;Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac;Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Zomepirac Sodium.

In any of the methods described herein, the cells can be administeredeither per se or, preferably as a part of a pharmaceutical compositionthat further comprises a pharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofthe adherent cells of the invention (i.e., adherent cells from placentaor adipose tissue, which are obtained from a 2D or 3D culture), withother chemical components such as pharmaceutically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of the cells to a subject.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

According to a preferred embodiment of the invention, the pharmaceuticalcarrier is an aqueous solution of saline.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

One may administer the pharmaceutical composition in a systemic manner(as detailed hereinabove). Alternatively, one may administer thepharmaceutical composition locally, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the invention may be manufactured byprocesses well known in the art, e.g., by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the inventionthus may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution,physiological salt buffer, or freezing medium containingcryopreservents. For transmucosal administration, penetrants appropriateto the barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. Preferably, a dose is formulated in ananimal model to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition, (see e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to levels of theactive ingredient which are sufficient to effectively regulate theneurotransmitter synthesis by the implanted cells. Dosages necessary toachieve the desired effect will depend on individual characteristics androute of administration. Detection assays can be used to determineplasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks ordiminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the individual being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc. The dosage and timing of administration willbe responsive to a careful and continuous monitoring of the individualchanging condition.

Models for inflammatory colon diseases include animal models ofulcerative colitis such as, but are not limited to, trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats and mice [Komori et al., JGastroenterol (2005) 40: 591-599; and Examples 4-5 hereinbelow].

Compositions including the preparation of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition.

Compositions of the invention may, if desired, be presented in a pack ordispenser device, such as an FDA approved kit, which may contain one ormore unit dosage forms containing the active ingredient. The pack may,for example, comprise metal or plastic foil, such as a blister pack. Thepack or dispenser device may be accompanied by instructions foradministration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

The adherent cells of the invention can be suitably formulated aspharmaceutical compositions which can be suitably packaged as an articleof manufacture. Such an article of manufacture comprises a label for usein treating ulcerative colitis or Crohn's disease, the packagingmaterial packaging a pharmaceutically effective amount of adherent cellsfrom a placenta or adipose tissue.

It will be appreciated that the article of manufacture may furthercomprise additional drugs for the treatment of colon inflammatoryconditions including, for example, anti-inflammatory agents,immunomodulatory agents, anti-inflammatory agents, and other drugs forthe treatment of inflammatory colon conditions (as described in furtherdetail hereinabove).

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 Methods of Generating Placenta Derived 3D Adherent Cells

Adherent cells were produced as was previously described (seeWO/2007/108003) in a bioreactor system containing 3D carriers to produce3D-adherent cells (designated herein as PLX).

Materials and Experimental Procedures

Placenta Derived Adherent Cells

Inner parts of a full-term delivery placenta (Bnei Zion medical center,Haifa, Israel) were cut under sterile conditions, washed 3 times withHank's Buffer and incubated for 3 hours at 37° C. with 0.1% Collagenase(1 mg/ml tissue; Sigma-Aldrich, St. Lewis, Mo.). Using gentle pipetting,suspended cells were then washed with DMEM supplemented with 10% FCS,Pen-Strep-Nystatin mixture (100 U/ml:100 μg/ml:1.25 un/ml) and 2 mML-glutamine, seeded in 75 cm² flasks and incubated at 37° C. in a tissueculture incubator under humidified condition with 5% CO₂.

Two Dimensional (2D) Cell Growth

Cells were allowed to adhere to a plastic surface for 72 hours afterwhich the media was changed every 3-4 days. After 2-3 passages, thecells were cryopreserved, thawed and seeded for a secondary growth inflasks. When reaching 60-80% confluence cells were detached from thegrowth flask using 0.25% trypsin-EDTA and seeded into new flasks(usually every 3-5 days), for another 2-5 passages. Cultured cells werethereafter collected for analysis or for culturing in bioreactors.

PluriX™ Plug Flow Bioreactor

The PluriX™ Plug Flow bioreactor (Pluristem, Haifa, Israel; see U.S.Pat. No. 6,911,201 and WO/2007/108003), was loaded with 1-100 ml packed3D porrosive carriers (4 mm in diameter) made of a non woven fabricmatrix of polyester. These carriers enable the propagation of large cellnumbers in a relatively small volume. Glassware was designed andmanufactured by Pluristem (Pluristem, Haifa, Israel). The bioreactor wasmaintained in an incubator of 37° C., with flow rate regulated andmonitored by a valve, and peristaltic pump. The bioreactor contains asampling and injection point, allowing the sequential seeding of cells.Culture medium was supplied at pH 6.7-7.4 from a reservoir. Thereservoir was supplied by a filtered gas mixture, containing air/CO₂/O₂at differing proportions, depending on cell density in the bioreactor.The O₂ proportion was suited to the level of dissolved O₂ at thebioreactor exit, determined by a monitor. The gas mixture was suppliedto the reservoir via silicone tubes or diffuser (Degania Bet, EmekHayarden, Israel). The culture medium was passed through a separatingcontainer which enables collection of circulating, nonadherent cells.Circulation of the medium was obtained by a peristaltic pump. Thebioreactor was further equipped with an additional sampling point andcontainers for continuous medium exchange.

Production of 3D-Adherent Cells (PLX)

Non-confluent primary human adherent 2D cell cultures, grown asdescribed above, were trypsinized, washed, resuspended in DMEMsupplemented with 10% FBS, Pen-Strep-Nystatin mixture (100 U/ml:100ug/ml:1.25 un/ml) and 2 mM L-glutamine, and seeded (10³-10⁵ cells/ml)via an injection point onto the 3D carriers in a sterile Plug Flowbioreactor. Prior to inoculation, bioreactor was filled with PBS-Ca—Mg(Biological Industries, Beit Ha'emek, Israel), autoclaved (120° C., 30min) and washed with Dulbecco's growth medium containing 10%heat-inactivated fetal calf serum and a Pen-Strep-Nystatin mixture (100U/ml:100 ug/ml:1.25 un/ml). Flow was kept at a rate of 0.1-5 ml/min.Seeding process involved cease of circulation for 2-48 hrs, therebyallowing the cells to settle on the carriers. Bioreactor was kept undercontrolled temperature (37° C.) and pH conditions (pH=6.7-7.4); using anincubator supplied with sterile air and CO₂ as needed. Growth medium wasreplaced 2-3 times a week. Circulation medium was replaced with freshDMEM media, every 4 hr to 7 days. At a density of 1×10⁶-1×10⁷ cells/ml(following 12-40 days of growth), total medium volume was removed fromthe bioreactor and bioreactor and carriers were washed 3-5 times withPBS. 3D-adherent cells were then detached from the carriers withTrypsin-EDTA; (Biological Industries, Beit Ha'emek, Israel; 3-15 minuteswith gentle agitation, 1-5 times), and were thereafter resuspended inDMEM and cryopreserved.

Example 2 Methods of Generating 2D Adherent Cells Suitable for Use inAccordance with the Present Teachings and the 2D Adherent CellsGenerated Thereby

2D adherent cells were produced which exhibit different characteristicsthen the above described 3D adherent cells (PLX, Example 1). Next, 2Dadherent cells from bone marrow or placenta origin were grown underosteocyte or adipocyte differentiation stimulating conditions.

Materials and Experimental Procedures

Manufacturing Process of 2D Adherent Cells

Receipt of Human Tissue

All placentas obtained were received from the maternity ward underapproval of the Helsinki Committee of the medical facility. Accordingly,all placenta donors signed an informed consent and Donor Screening andDonor Testing was performed (IPC1). Immediately after taking theplacenta from the donor (during the caesarean procedure), it was placedin a sterile plastic bag and then in a Styrofoam box with ice packs. Theplacenta was delivered and immediately placed in a quarantine area untilreleased to use by Quality Control (QC) and Quality Assurance (QA). Allthe following production steps were performed in a quarantine, cleanroom facility until QC approval of mycoplasma test results arrived andthe cells were release for 2D cell growth.

Recovery and Processing of Adherent Cells

To initiate the process, the placenta was cut into pieces under asepticconditions under laminar flow hood, washed with Hank's buffer solutionand incubated for 3 hours at 37° C. with 0.1% Collagenase (1 mgCollagenase/ml tissue). 2D cell medium (2D-Medium comprising DMEMsupplemented with 10% FBS, fungizone 0.25 μg/ml and gentamycine 50μg/ml) was added and the digested tissue was roughly filtered through asterile metal strainer, collected in a sterile beaker and centrifuged(10 minutes, 1200 RPM, 4° C.). Using gentle pipeting, suspended cellswere then washed with 2D-Medium supplemented with antibiotics, seeded in80 cm² flasks and incubated at 37° C. in a tissue culture incubatorunder humidified condition supplemented with 5% CO₂-Following 2-3 days,in which the cells were allowed to adhere to the flask surface, theywere washed with PBS and 2D-Medium was added.

Two Dimensional (2D) Cell Growth

Prior to the first passage, growth medium samples of 10% of the totalflask number in quarantine was pooled and taken for mycoplasma testing(IPC2). If cells were found to be negative for Mycoplasma (EZ-PCRMycoplasma kit, Biological Industries, Israel), cells were released fromquarantine. After 1-2 additional passages, cells were transferred to the2D production clean room (2DP). Once in Room 2DP, culture was continuedfor another 3-5 passages (of note, cells were grown in 2D-Mediumsupplemented with antibiotics until passage 2, thereafter cells weregrown in 2D-Medium without antibiotics). IPC-3 sample was taken forimmune phenotype after passage 4. Throughout the process, cultures weregrown in a tissue culture incubator under humidified conditions with 5%CO2 at 37° C. After a total of 6-8 passages (9-16 cell doublings), cellswere collected and cryopreserved as the 2D-Cell Stock (2DCS).

The first passage was usually carried out after 10-15 days. Beginning atpassage 2 and continuing until passage 6-8, cells were passaged when theculture reached 70-80% confluence, usually after 3-5 days (1.5-2doublings). The cells were detached from the flasks using 0.25%trypsin-EDTA (4 minutes at 37° C.) and seeded in a culture density of3±0.2×10³ cells/cm². The size of the tissue culture flasks raised as thepassages proceed. The culturing process started in 80 cm² tissue cultureflask, continued in 175 cm², then in 500 cm² (Triple flask) and finallythe cells were seeded into Cell Factory 10 tray (6320 cm²).

Prior to cryopreservation, at the end of 2DCS growth period, the growthmedium was collected and the sample was prepared to be sent to anapproved GLP laboratory for Mycoplasma test (IPC 4).

Cryopreservation Procedure for 2D-Cell-Stock Product

For 2DCS cryopreservation, 2D-cultured cells were collected underaseptic conditions using 0.25% trypsin-EDTA. The cells were centrifuged(1200 RPM, 10′, 4° C.), counted and re-suspended in 2D-Medium.

For freezing, cell suspensions were diluted. 1:1 with 2D-FreezingMixture (final concentrations was 10% DMSO, 40% FBS and 50% 2D-Medium).Approximately 1.5-2.5×10⁹ cells were manufactured from one placenta. 4ml of the cells were stored at a final concentration of 10×10⁶/ml in 5ml cryopreservation polypropylene vials. The vials were labeled andtransferred to a controlled rate freezer for a graduated temperaturereducing process (1° C./min), after which they were transferred tostorage in gas-phase of a liquid nitrogen freezer located in the ColdStorage Room. This material was referred to as the 2D-Cell Stock (2DCS)batch.

Cell Cycle Analysis

2D adherent cells and PLX cells were fixed with 70% EtOH O.N,centrifuged and re-suspended in a Propidium Iodide (PI) solutioncontaining 2 μg/ml PI (Sigma), 0.2 mg/ml Rnase A (Sigma) and 0.1% (v/v)Triton (Sigma) for 30 minutes. Cell cycle was analyzed by FACS.

Gene Expression Array (Microarray)

Adherent cells were obtained from human full term placentas and wereexpanded by 2D cultures or according to the teachings of WO/2007/108003(as described in detail in Examples 1-2). Three different batches ofcells were obtained from each of the expansion methods for furtherexamination.

RNA was extracted from the cells (Qiagen-Rneasy micro kit) and appliedto an Affymetrix whole genome expression array GeneChip® Human Exon 1.0ST Array (Affymetrix, Santa Clara, Calif., USA).

FACS Analysis of Membrane Markers

Cells were stained with monoclonal antibodies as previously described.In short, 400,000-600,000 cells were suspended in 0.1 ml flow cytometerbuffer in a 5 ml test tube and incubated for 15 minutes at roomtemperature (RT), in the dark, with each of the following monoclonalantibodies (MAbs): FITC-conjugated anti-human CD29 MAb (eBioscience), PEconjugated anti human CD73 MAb (Becton Dickinson), PE conjugated antihuman CD105 MAb (eBioscience), PE conjugated anti human CD90 MAb (BectonDickinson), FITC-conjugated anti-human CD45 MAb (IQProducts),PE-conjugated anti-human CD19 MAb (IQProducts), PE conjugated anti humanCD14 MAb (IQProducts), FITC conjugated anti human HLA-DR MAb(IQProduct), PE conjugated anti human CD34 MAb (IQProducts), FITCconjugated anti human CD31 MAb (eBioscience), FITC conjugated anti humanKDR MAb (R&D systems), anti human fibroblasts marker (D7-FIB)MAb(ACRIS), FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugatedanti-human CD86 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD),FITC-conjugated anti-human HLA-ABC MAb (BD), Isotype IgG1 FITCconjugated (IQ Products), Isotype IgG1 PE conjugated (IQ Products).

Cells were washed twice with flow cytometer buffer, resuspended in 500μl flow cytometer buffer and analyzed by flow cytometry using FC-500Flow Cytometer (Beckman Coulter). Negative controls were prepared withrelevant isotype fluorescence molecules.

Immunomodulation Assay

Human derived mononuclear cells (MNCs) were isolated from peripheralblood. Suspension of 200,000 MNCs per 200 μl medium (RPMI 1640 mediumcontaining 20% FBS per 96 well) were stimulated with 10 μg PHA/ml(SIGMA) in the presence of 20,000 2D adherent cells for 5 days underhumidified 5% CO₂ at 37° C. Four different batches of 2D adherent cellswere used. Three replicates of each group were seeded in 96-well plated.During the last 18 hrs of the 5-day culture, cells were pulsed with 1 μC³H-thymidine and further harvested over fiberglass filter. Thymidineuptake was quantified by a scintillation counter.

Induction of Osteogenesis in 2D Adherent Cells

Osteogenesis was carried out according to Chemicon osteogenesis kit (catno. scr028, Millipore, MA, USA)

Osteogenesis Induction Medium

Osteogenesis induction medium was freshly made prior to each mediumexchange using the kit components (See Table 1, below).

TABLE 1 Osteogenesis medium components Stock Component concentrationAmount Final con DMEM low glucose 8.7 ml 87% (Invitrogen, Gibco) Serum(heat inactivated) 1 ml 10% dexamethasone 1 mM 1 μl 0.1 μM AsorbicAcid-2- 0.1 M 20 μl 0.2 mM Phosphate solution Glycerol-2-Phosphate 1 M100 μL 10 Mm Solution L-glutamine ×100 100 μl ×1 Pen & Strep ×100 100 μl×1

To arrive at 1 mM dexamethasone solution, 900 μl ethanol was added to100 μl dexamethasone 10 mM solution. Stock solution was stored with therest of the kit's components at −20° C. A 50 ml serum vial was heatinactivated, divided into 5 ml aliquots and kept at −20° C. until use.

Coating 24-Well Tissue Culture Plates

A coating mixture comprising 12 μg/ml vitronectin and 12 μg/ml collagen(both included in the kit) was prepared by diluting each with 1×PBS.

The coating mixture was then added to the wells to cover the wellsurfaces (5 wells×2 plates were prepared). Plates were incubatedovernight at room temperature. The coating mixture was then removed andthe wells were rinsed once with PBS. Plates were aspirated right beforeuse.

Cell Growth

Placenta derived cells (plc11-3-1) or bone marrow derived cells (BM108)were plated (200,000 cells per well) in 1 ml growth medium comprisingDMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine(Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/mlFungizone (Invitrogen, Gibco). Placenta derived cells (4 wells×2 plates)or bone marrow derived cells (1 well×2 plates) were grown until 100%confluent (typically overnight) before initiating osteogenicdifferentiation.

When cells reached 100% confluence, growth medium was aspirated andreplaced with 1 ml osteogenesis induction medium (differentiation day1). Osteogenesis induction medium was replaced with fresh medium every2-3 days for a total of 14-17 days.

As a control, one of the two plates (for each of the cell types) was notincubated with osteogenesis differentiation medium but rather with thegrowth medium (described hereinabove).

On day 17, osteocytes were fixed and stained with Alizarin Red Solutionas depicted in detail below.

Staining Protocol

Osteocyte staining was performed by first carefully aspirating themedium from each well (carefully as to not aspirate the cells). Cellswere then fixed by incubating in iced cold 70% ethanol for 1 hour atroom temperature. The alcohol was then carefully aspirated and the cellswere rinsed twice with water (5-10 minutes each wash). The water wasthen aspirated and alizarin red solution (500-1000 μl) was added to thecells. Cells were incubated with alizarin red solution at roomtemperature for 30 minutes. Alizarin red was removed and the cells werewashed 4 times with 1 ml water and aspirated after each wash. Finally,1-1.5 ml water was added to each well to prevent cell drying. The plateswere microscopically visualized by an inverted Nikon microscope.

Induction of Osteogenesis in Modified Osteogenesis Induction Medium (2DAdherent Cells)

Osteogenesis induction medium was freshly made prior to each mediumexchange using the components listed in Table 2, below, along withVitamin D.

TABLE 2 Osteogenesis medium components Component Stock con Amount Finalcon DMEM high glucose (Biological 8.7 ml 87% Industries, Bet Haemek,Israel) L-glutamine ×100 100 μl ×1 Serum (heat inactivated) 1 ml 10%Dexamethasone (Chemicon) 10 mM 10 μl 10 μM Asorbic Acid-2-Phosphatesolution 0.1 M 20 μl 0.2 mM (Chemicon) Glycerol-2-Phosphate Solution 1 M100 μL 10 Mm (Chemicon) Vitamin D (Sigma) 10 μM 10 μL 10 nM Gentamycin(Biological Industries, ×100 100 μl ×1 Bet Haemek, Israel)

A 50 ml serum vial was heat inactivated, divided into 5 ml aliquots andkept at −20° C. until use.

Coating 48-Well Tissue Culture Plates

A coating mixture comprising 12 μg/ml vitronectin and 12 μg/ml collagen(both from Chemicon) was prepared by diluting each with 1×PBS.

The coating mixture was then added to the wells to cover the wellsurfaces (5 wells×2 plates were prepared). Plates were incubatedovernight at room temperature. The coating mixture was then removed andthe wells were rinsed once with PBS. Plates were aspirated right beforeuse.

Cell Growth

Placenta derived cells (PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1 fetalcells) were plated (100,000 cells per well) in 0.5 ml growth mediumcomprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 MmL-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and0.25 μg/ml Fungizone (Invitrogen, Gibco) (4 wells×2 plates). Bone marrowderived cells (BM109) were plated (150,000 cells per well) in 0.5 mlgrowth medium (as described above) (1 well×2 plates). Cells were grownuntil 100% confluent (typically overnight) before initiating osteogenicdifferentiation.

When cells reached 100% confluence, growth medium was aspirated andreplaced with 0.5 ml osteogenesis induction medium (differentiation day1). Osteogenesis induction medium was replaced with fresh medium every2-3 days for a total of 26 days.

As a control, one of the two plates (for each of the cell types) was notincubated with osteogenesis differentiation medium but rather with thegrowth medium (described hereinabove).

On day 26, osteocytes were fixed and stained with Alizarin Red Solutionas depicted in detail below.

Staining Protocol

Osteocyte staining was performed by first carefully aspirating themedium from each well (carefully as to not aspirate the cells). Cellswere then fixed by incubating in iced cold 70% ethanol for 1 hour atroom temperature. The alcohol was then carefully aspirated and the cellswere rinsed twice with water (5-10 minutes each wash). The water wasthen aspirated and alizarin red solution (500-1000 μl) was added to thecells. Cells were incubated with alizarin red solution at roomtemperature for 30 minutes. Alizarin red was removed and the cells werewashed 4 times with 1 ml water and aspirated after each wash. Finally,1-1.5 ml water was added to each well to prevent cell drying. The plateswere microscopically visualized by an inverted Nikon microscope.

Induction of Adipogenesis in 2D Adherent Cells

Adipogenesis was carried out according to Chemicon adipogenesis kit(Chemicon adipogenesis kit, cat no. scr020, Millipore, MA, USA)

Adipogenesis Induction Medium

Adipogenesis induction or maintenance mediums were freshly preparedprior to every medium exchange using the components depicted in Tables 3and 4, below.

TABLE 3 Adipogenesis induction medium components Component Stock conAmount Final con DMEM low glucose (Biological 4.4 ml 90% Industries, BetHaemek, Israel) Serum (heat inactivated) 0.5 ml 10% Dexamethasone(Sigma) 10 mM 0.5 μl 1 μM IBMX (Sigma) 0.5 M 5 μl 0.5 mM Insulin (Sigma)10 mg/ml 5 μL 10 μg/ml Indomethacin (Sigma) 10 mM 50 μl 100 μM Pen &Strep ×100 50 μl ×1

TABLE 4 Adipogenesis maintenance medium components Component Stock conAmount Final con DMEM low glucose 4.4 ml 90% Serum (heat inactivated)0.5 ml 10% Insulin 10 mg/ml 5 μL 10 μg/ml Pen & Strep ×100 50 μl ×1

Cell Growth

Placenta derived cells (plc11-3-1) or bone marrow derived cells (BM108)were plated (200,000 cells per well) in 1 ml growth medium comprisingDMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 Mm L-glutamine(Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and 0.25 μg/mlFungizone (Invitrogen, Gibco). Placenta derived cells (4 wells×2 plates)or bone marrow derived cells (1 well×2 plates) were grown until 100%confluent (typically overnight) before initiating adipogenesisdifferentiation.

When cells reached 100% confluence, growth medium was aspirated andreplaced with 1 ml adipogenesis induction medium (differentiation day1). Adipogenesis induction medium was replaced with fresh medium every2-3 days for a total of 25 days (as depicted in detail in Table 5,hereinbelow). Of note, monolayers of adipogenic cells were extremelyfragile and could easily dislodged from plates, therefore, mediumchanges were performed with gentle medium changes to avoid disruption ofthe lipid droplets.

As a control, one of the two plates (for each of the cell types) was notincubated with adipogenesis differentiation medium but rather with thegrowth medium (described hereinabove).

TABLE 5 Adipogenesis differentiation schedule Day Medium  1 AdipogenesisInduction medium  3 Adipogenesis Induction medium  5 AdipogenesisInduction medium  7 Adipogenesis Maintenance medium  9 AdipogenesisInduction medium 11 Adipogenesis Induction medium 13 AdipogenesisInduction medium 15 Adipogenesis Maintenance medium 17 AdipogenesisInduction medium 19 Adipogenesis Induction medium 21 AdipogenesisInduction medium

On day 25, adipocytes were fixed and stained with oil red solution asdepicted in detail below.

Staining Protocol

Adipocyte staining was performed by first carefully aspirating themedium from each well (carefully as to not aspirate the cells). Cellswere then fixed by incubating in 4% Para formaldehyde for 30-40 minutesat room temperature. The fixative was then carefully aspirated and thecells were rinsed three times with PBS (5-10 minutes each wash). Next,the PBS was aspirated and the cells were rinsed twice in water. Thewater was then aspirated and oil red solution (500-1000 μl) was added tothe cells. Cells were incubated with oil red solution at roomtemperature for 50 minutes. Oil red solution was removed and the cellswere washed 4 times with 1 ml water and aspirated after each wash.Finally, 1-1.5 ml water was added to each well to prevent cell drying.The plates were microscopically visualized by an inverted Nikonmicroscope.

Preparation of Oil Red Solution

Stock of 0.25 g oil red (Sigma) was used which was dissolved in 50 mliso-propanol by incubating 10-15 min in 37° C. bath.

For use, 30 ml of the stock stain was mixed with 20 ml DDW (left tostand for 10 minutes and then filtered with coffee filter paper). Theoil red solution was prepared fresh for each use.

Induction of Adipogenesis in Modified Adipogenesis Induction Medium (2DAdherent Cells)

Adipogenesis induction medium was freshly prepared prior to every mediumexchange using the components depicted in Table 6, below.

TABLE 6 Adipogenesis induction medium components Component Stock conAmount Final con DMEM low glucose 4.4 ml 90% Serum (heat inactivated)0.5 ml 10% Dexamethasone (Sigma) 1 mM 5 μl 1 μM IBMX (Sigma) 0.5 M 5 μl0.5 mM Insulin (Sigma) 10 mg/ml 5 μL 10 μg/ml Indomethacin (Sigma) 10 mM200 μl 100 μM Gentamycine (Biological Industries) 10 μl

Cell Growth

Placenta derived cells (PLC 8-2-1, PLC 15 3-4-2 or PLC 19-4-3-1 fetalcells) were plated (100,000 cells per well) in 0.5 ml growth mediumcomprising DMEM (Invitrogen, Gibco), 10% FCS (Invitrogen, Gibco), 2 MmL-glutamine (Sigma-Aldrich), 45 μg/ml Gentamicin-IKA (Teva Medical) and0.25 μg/ml Fungizone (Invitrogen, Gibco) (5 wells×2 plates).

Bone marrow derived cells (BM109) were plated (100,000 cells per well)in 0.5 ml growth medium comprising DMEM (Invitrogen, Gibco), 10% FCS(Invitrogen, Gibco), 2 Mm L-glutamine (Sigma-Aldrich), 45 μg/mlGentamicin-IKA (Teva Medical) and 0.25 μg/ml Fungizone (Invitrogen,Gibco) (4 well×2 plates). Cells were grown until 100% confluent(typically overnight) before initiating adipogenesis differentiation.

When cells reached 100% confluence, growth medium was aspirated andreplaced with 0.5 ml adipogenesis induction medium (differentiation day1). Adipogenesis induction medium was replaced with fresh medium every2-3 days for a total of 3-4 weeks.

As a control, one of the two plates (for each of the cell types) was notincubated with adipogenesis differentiation medium but rather with thegrowth medium (described hereinabove).

On day 26, adipocytes were fixed and stained with oil red solution asdepicted in detail below.

Staining Protocol

Adipocyte staining was performed by first carefully aspirating themedium from each well (carefully as to not aspirate the cells). Cellswere then fixed by incubating in 4% Para formaldehyde for 30-40 minutesat room temperature. The fixative was then carefully aspirated and thecells were rinsed three times with PBS (5-10 minutes each wash). Next,the PBS was aspirated and the cells were rinsed twice in water. Thewater was then aspirated and oil red solution (500-1000 μl) was added tothe cells. Cells were incubated with oil red solution at roomtemperature for 50 minutes. Oil red solution was removed and the cellswere washed 3 times with 1 ml double distilled water and aspirated aftereach wash. Finally, 1-1.5 ml water was added to each well to preventcell drying. The plates were microscopically visualized by an invertedNikon microscope.

Preparation of Oil Red Solution

Stock of 0.25 g oil red (Sigma) was used which was dissolved in 50 mliso-propanol by incubating 10-15 min in 37° C. bath.

For use, 30 ml of the stock stain was mixed with 20 ml DDW (left tostand for 10 minutes and then filtered with coffee filter paper). Theoil red solution was prepared fresh for each use.

Results

As is illustrated in Table 7, below, processing of the 2D adherent cellssuitable for use according to the present teachings differed from the 2Dstage of PLX (WO/2007/108003) in a few aspects. First, the new 2Dadherent cell's culture medium was supplemented with antibiotics onlyduring the initial culturing stage (up to passage 2). Also, the new 2Dadherent cells were cryopreserved only after 5-8 passages (i.e. at theend of culture) and not, as in the PLX process, during intermediatestages of 2D growth.

TABLE 7 Comparison of the 2 D adherent cells suitable for use accordingto the present teachings to those produced for PLX in WO/2007/108003 2 Dadherent cells of the Parameter WO/2007/108003 present teachings Tissueculture flask 80 cm² and 175 cm² 175 cm², triple flasks and Multi TrayMedium In all stages of the Up to passage 2 (included) supplemented withprocess antibiotics Cryopreservation of After 2-3 passages, After 5-8passages, then 2DCS then cryopreserved, cryopreserved and thawed thawedand seeded prior to use for a secondary growth in flasks for 2-5passages, prior to seeding in bioreactor Freezing container 2 mlcryogenic vials 5 ml cryogenic vials Freezing volume 1-1.5 ml 4 mlFreezing method Freezing container Controlled rate freezer (containsisopropyl alcohol)

Changes in the manufacturing process of the new 2D adherent cellsresulted in changes in characteristics of the obtained cells. Thesedifferences are summarized hereinbelow.

Cell cycle analysis of 2D adherent cells compared to 3D adherent cellsof WO/2007/108003—2D adherent cells were compared to 3D adherent cellsin order to examine the distribution of the cells between the differentphases of the cell cycle. As is clear from FIGS. 1A-B, 2D adherent cellsexhibited typical proliferating profile (distribution of cells betweenthe different phases of cell cycle). Specifically, 28% of cells were inS and G2/M phases (FIG. 1A). These results indicated that cells wereharvested during proliferation and that the culturing conditionssupported cell growth.

Conversely, 3D adherent cells exhibited lower rates of proliferatingcells. Less than 8% of cells were in S and G2/M phases (FIG. 1B). Theseresults indicated that cells were harvested while low levels ofproliferation were taking place and suggest that conditions in thebioreactor were suboptimal to support cell growth.

Microarray comparison between 2D cells suitable for use according to thepresent teachings to those obtained by the teachings ofWO/2007/108003—gene expression arrays enabled to simultaneously monitorgenome-wide expression profiles of adherent cells derived from humanfull term placentas expanded by 2D cultures or according to theteachings of WO/2007/108003 (PLX, see Example 1, hereinabove). Theseresults enabled to asses the molecular mechanism underlying phenotypicvariation between cells obtained by these different growth methods (seeTable 8, below).

TABLE 8 Gene expression in 2 D adherent cells suitable for use accordingto the present teachings compared to those expressed by PLX ofWO/2007/108003 2 D vs. Plurix (fold p-value Gene change) (treat)interferon-induced protein with tetratricopeptide repeats 21.820.0401812 leukocyte-derived arginine aminopeptidase 14.56 3.88E−06signal peptide, CUB domain, EGF-like 3 10.82 0.0255115 dickkopf homolog1 (Xenopus laevis) 6.84 3.06E−07 integrin, alpha 6 6.76 0.0411667keratin 27 pseudogene 27 6.39 0.000224998 similar to Keratin, type Icytoskeletal 18 (Cytokerati 6.24 0.000304949 aldehyde dehydrogenase 1family, member A1 5.84 0.00145807 G protein-coupled receptor, family C,group 5, member A 5.75 3.39E−05 coagulation factor III (thromboplastin,tissue factor) 5.55 0.012192 cyclin-dependent kinase inhibitor 3(CDK2-associated 5.51 0.000732492 dual G protein-coupled receptor 1265.50 0.00197635 DEP domain containing 1 5.41 0.000370513 SHC SH2-domainbinding protein 1 4.96 0.00430878 centrosomal protein 55kDa 4.780.0021952 interferon-induced protein with tetratricopeptide repeats 4.660.0139777 NUF2, NDC80 kinetochore complex component, homolog 4.610.00276524 (S. cere mal, T-cell differentiation protein-like 4.440.00664216 interferon-induced protein with tetratricopeptide repea 4.420.00357376 kinesin family member 18A 4.33 0.00134108 cholinergicreceptor, muscarinic 2 4.07 0.0320078 cell division cycle 2, G1 to S andG2 to M 4.06 0.0017111 non-SMC condensin I complex, subunit G 4.060.00537097 denticleless homolog (Drosophila) 4.06 0.00141153shugoshin-like 1 (S. pombe) 4.00 0.00101318 chromosome 13 open readingframe 3 3.98 0.000548296 PDZ binding kinase 3.97 0.00784983 lymphocytecytosolic protein 1 (L-plastin) 3.97 0.0049584 WAS 3.96 0.00178153cyclin E2 3.94 0.000203389 cathepsin C 3.93 0.00532262 integrin, alpha 4(antigen CD49D, alpha 4 subunit of 3.91 0.0158411 VLA-4 KIAA0101 3.900.0105909 kinesin family member 20A 3.90 0.00582352 opioid growth factorreceptor-like 1 3.87 0.00114551 anillin, actin binding protein 3.830.010923 catenin (cadherin-associated protein), alpha-like 1 3.767.46E−05 cell division cycle 20 homolog (S. cerevisiae) 3.70 0.00514206diaphanous homolog 3 (Drosophila) 3.69 0.00107709 family with sequencesimilarity 111, member B 3.69 0.000125819 aurora kinase A 3.660.00632571 fibroblast growth factor 7 (keratinocyte growth factor) 3.640.0328983 maternal embryonic leucine zipper kinase 3.63 0.00908391 RhoGDP dissociation inhibitor (GDI) beta 3.63 0.00200066 centromere proteinN 3.62 0.000540143 MAD2 mitotic arrest deficient-like 1 (yeast) 3.620.00488102 thymidylate synthetase 3.61 0.00685584 cyclin B2 3.600.016544 regulator of G-protein signalling 4 3.59 0.00781061 chromosome6 open reading frame 173 3.58 0.00222408 hyaluronan-mediated motilityreceptor (RHAMM) 3.55 0.00467816 BUB1 budding uninhibited bybenzimidazoles 1 homolog 3.54 0.0108258 (yeast SPC25, NDC80 kinetochorecomplex component, 3.53 0.00568662 homolog (S. ce establishment ofcohesion 1 homolog 2 (S. cerevisiae) 3.52 0.000773033 cyclin A2 3.510.00965934 CDC28 protein kinase regulatory subunit 2 3.51 0.0128024keratin 18 3.47 0.000514523 ribonucleotide reductase M2 polypeptide 3.460.00834059 arylacetamide deacetylase-like 1 3.44 0.000902645 kinesinfamily member 11 3.43 0.00915145 Rho GTPase activating protein 11A 3.410.00834174 GINS complex subunit 1 (Psf1 homolog) 3.39 0.00104515 discs,large homolog 7 (Drosophila) 3.38 0.0317074 TTK protein kinase 3.380.0112171 deleted in lymphocytic leukemia, 2 3.38 0.0109528 replicationfactor C (activator 1) 3, 38kDa 3.37 0.00109668 solute carrier family 7,(cationic amino acid transporte 3.36 0.00688017 dual-specificitytyrosine-(Y)-phosphorylation regulated ki 3.34 0.0234606 kinesin familymember 2C 3.34 0.0059888 heat shock 22kDa protein 8 3.32 0.0219583polo-like kinase 1 (Drosophila) 3.30 0.0140309 v-myb myeloblastosisviral oncogene homolog (avian)-lik 3.28 0.0043878 trypsinogen C 3.280.00416276 thymidine kinase 1, soluble 3.27 0.00124134 NAD(P)Hdehydrogenase, quinone 1 3.27 0.000282423 high-mobility group box 2 3.240.0196872 cell division cycle associated 2 3.24 0.0122226 apolipoproteinB mRNA editing enzyme, catalytic 3.23 0.00308692 polypep serpinpeptidase inhibitor, clade B (ovalbumin), member 3.22 0.0190218 guaninenucleotide binding protein (G protein), gamma 11 3.22 0.00140559chromosome 15 open reading frame 23 3.21 0.000147331 kinesin familymember 14 3.19 0.00947901 transmembrane protein 154 3.18 0.0045589glycerol kinase 3.16 2.66E−05 KIAA1524 3.15 0.0380688 coagulation factorXIII, B polypeptide 3.14 0.0294465 tight junction protein 2 (zonaoccludens 2) 3.13 0.00012562 nei endonuclease VIII-like 3 (E. coli) 3.120.00115606 pleckstrin 2 3.11 0.0304429 kinesin family member 23 3.090.00790585 Rac GTPase activating protein 1 3.09 0.00381613 keratinocytegrowth factor-like protein 1 3.07 0.0300588 keratinocyte growthfactor-like protein 1 3.07 0.0300588 keratinocyte growth factor-likeprotein 1 3.07 0.0300588 transcription factor 19 (SC1) 3.07 0.00109627OCIA domain containing 2 3.07 0.00122147 lung cancermetastasis-associated protein 3.06 0.00148024 transcription factor 19(SC1) 3.05 0.00124327 transcription factor 19 (SC1) 3.05 0.00124327 RhoGTPase activating protein 29 3.05 0.0466211 glucosaminyl (N-acetyl)transferase 1, core 2 (beta-1,6-N- 3.05 0.0197148 replication factor C(activator 1) 4, 37kDa 3.04 0.00164152 protein regulator of cytokinesis1 3.01 0.0325664 transforming, acidic coiled-coil containing protein 32.98 0.0014577 cancer susceptibility candidate 5 2.96 0.0330594nucleolar and spindle associated protein 1 2.96 0.00520875 cyclin B12.96 0.0103092 transmembrane protein 48 2.96 0.00458248 ZW10 interactor2.95 1.88E−05 endonuclease domain containing 1 2.95 0.000429245hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan 2.94 0.000634057synd fucosidase, alpha-L-2, plasma 2.94 0.00540929 ubiquitin-conjugatingenzyme E2T (putative) 2.93 0.00741886 lipase A, lysosomal acid,cholesterol esterase (Wolman 2.92 0.0167385 dise villin 2 (ezrin) 2.920.0131934 glycerol kinase 2.90 3.37E−06 WD repeat domain 76 2.890.0023531 CD97 molecule 2.89 0.00994045 chromosome 18 open reading frame24 2.89 0.00347442 topoisomerase (DNA) II alpha 170kDa 2.89 0.0321109integrin, alpha 3 (antigen CD49C, alpha 3 subunit of 2.87 0.00574148VLA-3 family with sequence similarity 29, member A 2.85 0.00111165kinesin family member 4A 2.85 0.0114203 BRCA1 associated RING domain 12.85 0.000540414 serum 2.84 0.0387246 RAD51 homolog (RecA homolog, E.coli) (S. cerevisiae) 2.83 0.000854739 Fanconi anemia, complementationgroup I 2.83 0.00464532 dihydrofolate reductase 2.82 0.00178879 claspinhomolog (Xenopus laevis) 2.81 0.00683624 ornithine decarboxylase 1 2.810.00144868 sperm associated antigen 5 2.80 0.00906321 histone cluster 1,H3b 2.80 0.0304598 ATPase family, AAA domain containing 2 2.790.00415258 KIAA0286 protein 2.79 0.00130563 guanine nucleotide bindingprotein (G protein), alpha inhi 2.76 0.00184597 BUB1 budding uninhibitedby benzimidazoles 1 homolog 2.74 0.0166047 beta dihydrofolate reductasepseudogene 2.74 0.00141306 brix domain containing 1 2.73 0.00471977cytoskeleton associated protein 2 2.72 0.0030499 mitochondrial ribosomalprotein S28 2.72 0.00298194 polymerase (DNA directed), epsilon 2 (p59subunit) 2.72 0.00479612 family with sequence similarity 72, member A2.72 0.00143248 EBNA1 binding protein 2 2.70 0.00296292 similar to 40Sribosomal protein SA (P40) (34 2.70 0.0385298 adiposedifferentiation-related protein 2.70 0.000331751 thioredoxin reductase 12.70 0.000197486 minichromosome maintenance complex component 5 2.690.00475504 von Hippel-Lindau binding protein 1 2.69 0.00329061 SCL 2.680.00390288 Fanconi anemia, complementation group D2 2.68 0.0281405 NIMA(never in mitosis gene a)-related kinase 2 2.68 0.00289469 PHD fingerprotein 19 2.68 0.000177604 microsomal glutathione S-transferase 1 2.680.041701 breast cancer 2, early onset 2.68 0.00586847 non-SMC condensinI complex, subunit H 2.67 0.0216752 chromosome 13 open reading frame 272.67 0.0234588 histone cluster 1, H2bg 2.67 0.000180822 non-SMCcondensin II complex, subunit G2 2.66 0.0130322 centromere protein I2.64 0.0106816 stomatin 2.64 0.00387095 glutathione S-transferase omega1 2.63 0.000648379 protein tyrosine phosphatase-like A domain containing2.62 0.0419644 calcyclin binding protein 2.62 0.00524566 KIT ligand 2.610.00641955 ubiquitin-conjugating enzyme E2L 3 2.61 0.00343347 serpinpeptidase inhibitor, clade B (ovalbumin), member 2.60 0.0030439 ATPase,Ca++ transporting, plasma membrane 4 2.60 0.023011 TPX2,microtubule-associated, homolog (Xenopus laevis) 2.60 0.0253137 thyroidhormone receptor interactor 13 2.59 0.0118319 H2A histone family, memberZ 2.59 0.0129697 CDC28 protein kinase regulatory subunit 1B 2.570.0107391 cell division cycle associated 3 2.57 0.006289 minichromosomemaintenance complex component 8 2.57 0.000841489 E2F transcriptionfactor 2 2.55 0.0496479 TIMELESS interacting protein 2.55 0.00771062minichromosome maintenance complex component 4 2.54 0.00342054 polo-likekinase 4 (Drosophila) 2.53 0.00209633 kinesin family member C1 2.530.00821937 dihydrofolate reductase 2.52 0.00307793 glycerol-3-phosphatedehydrogenase 2 (mitochondrial) 2.52 0.00211969 TGF beta-induciblenuclear protein 1 2.51 0.0365579 integrin, alpha 2 (CD49B, alpha 2subunit of VLA-2 2.51 0.0210165 receptor MLF1 interacting protein 2.510.0177203 heat shock 70kDa protein 2 2.50 0.0215102 hairy and enhancerof split 1, (Drosophila) 2.50 0.000283509 ATP-binding cassette,sub-family C (CFTR 2.49 0.00382491 serglycin 2.48 0.0443487 sema domain,immunoglobulin domain (Ig), short basic 2.47 0.008548 doma ankyrinrepeat domain 1 (cardiac muscle) 2.47 0.00911953 transporter 1,ATP-binding cassette, sub-family B (MDR 2.47 0.00859077 transporter 1,ATP-binding cassette, sub-family B (MDR 2.47 0.00859077 transporter 1,ATP-binding cassette, sub-family B (MDR 2.47 0.00859077 histone cluster1, Hib 2.46 0.0470898 family with sequence similarity 72, member A 2.460.00165234 membrane bound O-acyltransferase domain containing 1 2.460.01185 epidermal growth factor receptor pathway substrate 8 2.450.0194949 ASF1 anti-silencing function 1 homolog B (S. cerevisiae) 2.450.00543408 dedicator of cytokinesis 11 2.44 0.00697577 family withsequence similarity 72, member A 2.44 0.00162905 actin related protein 22.44 0.000288443 CTP synthase 2.43 8.80E−05 M-phase phosphoprotein 12.43 0.0271814 CDC28 protein kinase regulatory subunit 1B 2.43 0.0145263histone cluster 1, H2ai 2.43 0.0161621 high-mobility group nucleosomalbinding domain 2 2.42 0.0030536 heat shock 70kDa protein 1A 2.420.00734287 heat shock 70kDa protein 1A 2.42 0.00674816 carnitinepahnitoyltransferase 1A (liver) 2.41 0.00170894 neurofilament, mediumpolypeptide 150kDa 2.41 0.0190611 transmembrane protein 62 2.410.00761064 vaccinia related kinase 1 2.40 0.0233182 geminin, DNAreplication inhibitor 2.40 0.00167629 phosphoglucomutase 2 2.400.00818204 lamin B1 2.40 0.0477748 keratin 18 2.40 0.000112551 deafness,autosomal dominant 5 2.39 0.00235481 proteasome (prosome, macropain)subunit, beta type, 9 2.39 0.0202595 (lar proteasome (prosome,macropain) subunit, beta type, 9 2.39 0.0202595 (lar proteasome(prosome, macropain) subunit, beta type, 9 2.39 0.0202595 (larchromosome 12 open reading frame 31 2.39 0.0173089 isocitratedehydrogenase 3 (NAD+) alpha 2.39 0.00297129 forkhead box M1 2.380.0203154 transmembrane protein 106C 2.38 0.000214223 hypotheticalprotein LOC729012 2.38 0.000446087 PHD finger protein 1 2.37 0.010191mitochondrial ribosomal protein L15 2.37 0.0306092 elastin microfibrilinterfacer 2 2.37 0.0192072 hypothetical protein DKFZp762E1312 2.370.00726778 retinoblastoma-like 1 (p107) 2.36 0.00319946 tissue factorpathway inhibitor (lipoprotein-associated 2.36 0.0356227 epithelial celltransforming sequence 2 oncogene 2.36 0.000571152 crystallin, zeta(quinone reductase) 2.36 0.0370884 hect domain and RLD 4 2.36 0.00679184high-mobility group nucleosomal binding domain 2 2.36 0.00384071 celldivision cycle 25 homolog A (S. pombe) 2.36 0.000341692 thymopoietin2.35 0.0223176 interferon-induced protein with tetratricopeptide repeats2.34 0.0177928 Bloom syndrome 2.34 0.0209259 dual specificityphosphatase 1 2.34 0.00211272 elongation factor, RNA polymerase II, 22.34 0.0130017 small nuclear ribonucleoprotein D1 polypeptide 16kDa 2.340.0334665 CDC45 cell division cycle 45-like (S. cerevisiae) 2.330.00735977 exonuclease 1 2.33 0.00739393 ribosomal protein L39-like 2.330.00429384 histone cluster 1, H2bh 2.33 0.0377748 ribonucleotidereductase M1 polypeptide 2.33 0.000170076 sulfiredoxin 1 homolog (S.cerevisiae) 2.32 5.14E−05 multiple coagulation factor deficiency 2 2.310.0116892 proteasome (prosome, macropain) subunit, alpha type, 3 2.310.0195874 ribonuclease H2, subunit A 2.30 0.00669936 minichromosomemaintenance complex component 10 2.29 0.0037925 heat shock 70kDa protein1B 2.28 0.0048959 heat shock 70kDa protein 1B 2.28 0.0054404 heat shock70kDa protein 1B 2.28 0.0054404 ATPase, Na+ 2.28 0.000381464hypothetical protein LOC201725 2.28 0.000313319 cathepsin L1 2.270.0314419 cell division cycle associated 5 2.27 0.01021 RAB8B, memberRAS oncogene family 2.27 0.00417066 SPC24, NDC80 kinetochore complexcomponent, 2.27 0.00287227 homolog (S. ce gamma-glutamyl hydrolase(conjugase, 2.26 0.0195219 folylpolygammaglutamyl cell division cycle 25homolog C (S. pombe) 2.25 0.0169914 mutS homolog 2, colon cancer,nonpolyposis type 1 (E. 2.25 0.00578953 coli) metallothionein 1 L (gene.2.25 0.00709646 RRS1 ribosome biogenesis regulator homolog (S. 2.240.0120061 cerevisiae) cell division cycle associated 8 2.24 0.00619878shugoshin-like 2 (S. pombe) 2.24 0.000852557 mRNA turnover 4 homolog (S.cerevisiae) 2.24 0.00373104 ST6(alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 2.24 0.00830766 v-etserythroblastosis virus E26 oncogene homolog 2 2.23 0.0364123 (avian)replication factor C (activator 1) 2, 40kDa 2.23 0.00768959 NIMA (neverin mitosis gene a)-related kinase 7 2.23 0.00159114 basic leucine zipperand W2 domains 2 2.23 0.0190782 histone cluster 1, H2bf 2.23 0.0124279eukaryotic translation initiation factor 1A, X-linked 2.23 0.00330183transporter 1, ATP-binding cassette, sub-family B (MDR 2.22 0.0164234transporter 1, ATP-binding cassette, sub-family B (MDR 2.22 0.0164234transporter 1, ATP-binding cassette, sub-family B (MDR 2.22 0.0164234polymerase (RNA) III (DNA directed) polypeptide G 2.22 0.0298794 (32kD)phosphatidylinositol-4-phosphate 5-kinase, type II, alph 2.22 0.00964099proteasome (prosome, macropain) 26S subunit, ATPase, 6 2.22 0.024269pituitary tumor-transforming 1 2.21 0.0485166 histone cluster 2, H3d2.21 0.0102932 sulfide quinone reductase-like (yeast) 2.21 0.0473641serglycin 2.20 0.00880325 ribosomal protein L22-like 1 2.20 0.00335381membrane protein, palmitoylated 1, 55kDa 2.20 0.000396285 solute carrierfamily 24 (sodium 2.20 0.0328774 STAM binding protein-like 1 2.200.0181743 WD repeat and HMG-box DNA binding protein 1 2.20 0.0034833CSE1 chromosome segregation 1-like (yeast) 2.20 0.0013662 originrecognition complex, subunit 6 like (yeast) 2.20 0.00182466transcription factor A, mitochondrial 2.19 0.0110092 exosome component 82.19 0.00132017 mitochondrial ribosomal protein L1 2.19 0.0361058sphingomyelin synthase 2 2.19 0.0020701 deoxycytidine kinase 2.180.00101444 family with sequence similarity 29, member A 2.18 0.00469407chromosome 6 open reading frame 167 2.18 0.0011095 dual specificityphosphatase 11 (RNA 2.18 0.00426788 F-box protein 45 2.18 0.00510098ras-related C3 botulinum toxin substrate 2 (rho family, 2.17 0.0292466sma FK506 binding protein 5 2.17 0.0193805 breast cancer 1, early onset2.17 0.0180553 nuclear factor I 2.17 0.0010313 thioredoxin 2.17 0.009636SH2 domain containing 4A 2.16 0.0323646 TGF beta-inducible nuclearprotein 1 2.16 0.00285964 PSMC3 interacting protein 2.16 0.00766442chromosome 3 open reading frame 14 2.15 0.0377617 polycomb group ringfinger 5 2.15 0.000294142 centrosomal protein 27kDa 2.15 0.00931602family with sequence similarity 64, member A 2.14 0.0019785 acidic(leucine-rich) nuclear phosphoprotein 32 family, m. 2.14 0.0300263sterol O-acyltransferase (acyl-Coenzyme A: cholesterol 2.14 0.0193637acy TATA box binding protein (TBP)-associated factor, RNA 2.130.00514451 poly origin recognition complex, subunit 5-like (yeast) 2.130.049697 Rac GTPase activating protein 1 pseudogene 2.13 0.000269488LSM5 homolog, U6 small nuclear RNA associated (S. 2.13 0.00264664cerevisia minichromosome maintenance complex component 7 2.13 0.0457691met proto-oncogene (hepatocyte growth factor receptor) 2.13 0.0318147tripartite motif-containing 25 2.13 0.0456344 chromosome 13 open readingframe 34 2.13 0.000702936 patatin-like phospholipase domain containing 42.13 0.0168306 minichromosome maintenance complex component 6 2.120.0161279 intraflagellar transport 80 homolog (Chlamydomonas) 2.120.0247286 peptidylprolyl isomerase F (cyclophilin F) 2.12 0.00093846UTP15, U3 small nucleolar ribonucleoprotein, homolog 2.12 0.00482559 (S.c TAF9B RNA polymerase II, TATA box binding protein 2.12 0.0170365(TBP)-as TAF9B RNA polymerase II, TATA box binding protein 2.120.0170365 (TBP)-as ecotropic viral integration site 2B 2.12 0.01714083′-phosphoadenosine 5′-phosphosulfate synthase 2 2.12 1.43E−05proteasome (prosome, macropain) activator subunit 2 2.12 0.00609885(PA28 ADAM metallopeptidase with thrombospondin type 1 2.12 0.0102751motif, flap structure-specific endonuclease 1 2.12 0.006882 S100 calciumbinding protein A3 2.12 0.0324073 RAD18 homolog (S. cerevisiae) 2.110.0016685 minichromosome maintenance complex component 3 2.11 0.0018389exosome component 3 2.11 0.0249115 cysteinyl-tRNA synthetase 2,mitochondrial (putative) 2.11 0.00564558 glutamate-cysteine ligase,modifier subunit 2.11 0.00378868 brix domain containing 1 2.110.00981178 kinesin family member 22 2.11 0.0192406 UTP11-like, U3 smallnucleolar ribonucleoprotein, (yeast) 2.10 0.0132794 v-ral simianleukemia viral oncogene homolog B (ras 2.10 0.012225 related meioticnuclear divisions 1 homolog (S. cerevisiae) 2.10 0.00164447phenylalanyl-tRNA synthetase, beta subunit 2.10 0.000245973 similar toUbiquitin-conjugating enzyme E2S (Ubiqui 2.10 0.000415822 coiled-coildomain containing 68 2.10 0.00227586 lamin B receptor 2.10 0.000151784Niemann-Pick disease, type C1 2.10 0.0108117 hydroxysteroiddehydrogenase like 2 2.09 3.71E−05 RMI1, RecQ mediated genomeinstability 1, homolog (S. 2.09 0.00294705 cerev overexpressed in coloncarcinoma-1 2.09 0.015322 hypothetical protein FL120425 2.09 0.0174225primase, polypeptide 1, 49kDa 2.09 0.00801018 chromosome 20 open readingframe 121 2.09 0.0146323 microtubule associated serine 2.08 0.00536974endothelial differentiation, sphingolipid G-protein-coupled 2.080.0132848 homeobox A9 2.08 0.00520942 centromere protein L 2.080.000880856 nucleolar complex associated 3 homolog (S. cerevisiae) 2.070.000373346 fibroblast growth factor 7 (keratinocyte growth factor) 2.070.0173208 cysteine rich transmembrane BMP regulator 1 (chordin- 2.070.0267286 like) nucleoporin 155kDa 2.07 0.00218453 FLJ20105 protein 2.060.0127979 CD44 molecule (Indian blood group) 2.06 0.000651436 polymerase(DNA directed), alpha 2 (70kd) subunit) 2.06 0.0033903 v-mybmyeloblastosis viral oncogene homolog (avian)- 2.06 0.00989416 like 2origin recognition complex, subunit 1-like (yeast) 2.06 0.00207753hypothetical protein FLJ25416 2.06 0.000177531 kinesin family member 222.06 0.0242075 heat shock 60kDa protein 1 (chaperonin) 2.06 0.0327412minichromosome maintenance complex component 2 2.05 0.0021347fumarylacetoacetate hydrolase (fumarylacetoacetase) 2.05 3.88E−05glycerol kinase 3 pseudogene 2.05 0.0103203 retinitis pigmentosa 2(X-linked recessive) 2.05 0.0264185 U2AF homology motif (UHM) kinase 12.05 0.0255167 chaperonin containing TCP1, subunit 5 (epsilon) 2.040.00125909 ATPase, H+ transporting, lysosomal 34kDa, V1 subunit D 2.040.0317453 transcription termination factor, RNA polymerase II 2.040.000393489 succinate-CoA ligase, GDP-forming, beta subunit 2.040.0028167 cyclin-dependent kinase inhibitor 1B (p27, Kip1) 2.040.00183021 tyrosine 3-monooxygenase 2.04 0.00021508 cofactor requiredfor Sp1 transcriptional activation, subu 2.04 0.00141809glycosyltransferase 8 domain containing 3 2.03 0.022868 ribosomal RNAprocessing 15 homolog (S. cerevisiae) 2.03 0.0274884 glycogenin 1 2.030.0224317 hypothetical protein FLJ40869 2.03 0.00444509 proliferatingcell nuclear antigen 2.03 0.0031727 sterile alpha motif domaincontaining 12 2.03 0.0232188 chromosome 16 open reading frame 59 2.030.00185191 cofilin 2 (muscle) 2.03 0.0459235 eukaryotic translationinitiation factor 2, subunit 2 bet 2.03 0.0139947 chromatin assemblyfactor 1, subunit B (p60) 2.03 0.0119687 Zwilch, kinetochore associated,homolog (Drosophila) 2.02 0.000725107 ATP-binding cassette, sub-family E(OABP), member 1 2.02 0.00454751 LSM3 homolog, U6 small nuclear RNAassociated (S. 2.02 0.0199824 cerevisia IQ motif containing GTPaseactivating protein 3 2.02 0.0495882 tubulin, alpha 1c 2.02 0.00862586DBF4 homolog (S. cerevisiae) 2.01 0.0458795 amyloid beta precursorprotein binding protein 1 2.01 0.000910538 suppressor of variegation 3-9homolog 1 (Drosophila) 2.01 0.00224324 THO complex 7 homolog(Drosophila) 2.01 0.0047251 amyotrophic lateral sclerosis 2 (juvenile)chromosome re 2.01 0.0484466 nucleoporin 37kDa 2.01 0.00652747 nucleolarprotein 11 2.01 0.000852662 ATP synthase, H+ transporting, mitochondrialF0 complex 2.01 0.00866627 histone cluster 1, H2ai 2.01 0.0129155phytoceramidase, alkaline 2.01 0.0157729 primase, polypeptide 2A, 58kDa2.01 0.00290097 similar to High mobility group protein B1 (High mobili2.00 0.000363158 mastermind-like 3 (Drosophila) −2.00 0.00386667UDP-N-acetyl-alpha-D-galactosamine: polypeptide N- −2.01 0.0268634acetylga ring finger protein 122 −2.01 0.0236621 chromodomain helicaseDNA binding protein 3 −2.01 6.39E−05 centaurin, gamma-like family,member 10 pseudogene −2.01 8.70E−05 chromosome 7 open reading frame 10−2.01 0.00738442 chromosome 6 open reading frame 111 −2.01 0.0104492centaurin, gamma-like family, member 10 pseudogene −2.01 0.000334818Prader-Willi syndrome chromosome region 1 −2.01 0.0415526 KIAA1245 −2.010.0186309 peroxidasin homolog (Drosophila) −2.01 0.00219049 melanomaantigen family D, 4 −2.02 0.0263076 melanoma antigen family D, 4 −2.020.0263076 glucosidase, alpha; acid (Pompe disease, glycogen storage−2.02 0.000418401 di phospholipase A2 receptor 1, 180kDa −2.030.00069343 glycosyltransferase 8 domain containing 2 −2.03 0.0173546KIAA1546 −2.03 0.000255634 protocadherin beta 9 −2.03 0.0285124 TBC1domain family, member 3B −2.03 0.000414974 sushi, nidogen and EGF-likedomains 1 −2.03 0.00161129 microtubule-actin crosslinking factor 1 −2.040.00216 region containing neuroblastoma breakpoint family, −2.040.0213393 golgi autoantigen, golgin subfamily a-like pseudogene −2.040.0182674 transducin-like enhancer of split 4 (E(sp1) homolog, −2.040.0164153 Drosop solute carrier family 22 (organic cation transporter),−2.05 0.0137275 neighbor of Punc E11 −2.05 0.0184739 insulin-like growthfactor binding protein 5 −2.05 0.011614 KIAA1245 −2.06 0.0185376 vitaminD (1,25- dihydroxyvitamin D3) receptor −2.06 0.000192208 B-cell CLL−2.06 0.00343507 KIAA1305 −2.06 0.00813727 KIAA1245 −2.06 0.0185609centaurin, gamma-like family, member 10 pseudogene −2.07 3.08E−05 TBC1domain family, member 3B −2.07 0.00141297 similar to TBC1 domain familymember 3 (Rab GTPase- −2.08 0.00105098 mannosidase, alpha, class 2B,member 1 −2.08 0.000353303 cysteine-rich PAK1 inhibitor −2.080.000125336 midline 1 (Opitz −2.08 0.00130803 small nucleolar RNA, H−2.09 0.017124 urocortin 2 −2.09 0.00172263 neuroblastoma breakpointfamily, member 11 −2.09 0.0138065 collagen, type VI, alpha 3 −2.092.09E−06 neuroblastoma breakpoint family, member 11 −2.09 0.0148372hypothetical protein LOC646870 −2.09 0.0117625 calsyntenin 3 −2.090.00300887 cortactin binding protein 2 −2.09 2.28E−05 synaptic vesicleglycoprotein 2A −2.10 0.00704212 similar to Dynamin-1 (D100) (Dynamin,brain) (B-dyn −2.10 0.0190733 similar to Dynamin-1 (D100) (Dynamin,brain) (B-dyn −2.10 0.0190733 similar to TBC1 domain family member 3(Rab GTPase- −2.10 0.00108467 Notch homolog 2 (Drosophila) N-terminallike −2.10 0.0193058 matrix-remodelling associated 5 −2.11 0.000317637complement component 1, s subcomponent −2.11 0.0395863 cysteine sulfinicacid decarboxylase −2.11 0.00428211 hypothetical protein FLJ36144 −2.110.00958437 hypothetical protein FLJ36144 −2.11 0.00958437dihydropyrimidinase-like 3 −2.12 0.0165203 procollagen C-endopeptidaseenhancer −2.12 0.0039236 golgi autoantigen, golgin subfamily a-likepseudogene −2.12 0.00720508 TBC1 domain family, member 3B −2.120.00122924 collagen, type VII, alpha 1 (epidermolysis bullosa, dystr−2.13 0.00109233 versican −2.14 0.023885 mannose receptor, C type 2−2.14 0.00012142 golgi autoantigen, golgin subfamily a-like pseudogene−2.14 0.00767095 dynamin 1 −2.15 0.00139674 TBC1 domain family, member3B −2.16 0.00130459 PHD finger protein 21A −2.17 0.00980401 centaurin,gamma-like family, member 10 pseudogene −2.17 0.000180846 slit homolog 3(Drosophila) −2.17 0.02844 neuroepithelial cell transforming gene 1−2.18 0.0109689 cyclin L2 −2.18 0.00093459 similar to dJ402H5.2 (novelprotein similar to wo −2.18 0.00621503 phospholipase D family, member 3−2.18 1.17E−05 collagen, type VIII, alpha 1 −2.19 0.00187242 cyclin L2−2.19 0.00109621 protocadherin beta 14 −2.20 0.0103892 matrixmetallopeptidase 2 (gelatinase A, 72kDa gelatinase, −2.20 5.59E−05 lysyloxidase-like 4 −2.21 0.0120148 golgi autoantigen, golgin subfamilya-like pseudogene −2.21 0.00977719 WW domain containing transcriptionregulator 1 −2.21 0.0379899 PDZ domain containing RING finger 3 −2.210.00931014 chromosome 14 open reading frame 37 −2.21 0.0182453 brain andacute leukemia, cytoplasmic −2.22 0.0476919 calcium channel,voltage-dependent, L type, alpha 1C sub −2.22 0.0189661 jun oncogene−2.23 7.21E−05 interleukin 19 −2.23 0.0310328 centaurin, gamma-likefamily, member 10 pseudogene −2.23 0.000595086 centaurin, gamma-likefamily, member 10 pseudogene −2.23 0.000595086 — −2.24 0.00666187 golgiautoantigen, golgin subfamily b, macrogolgin (with −2.24 0.0164005chromosome 15 open reading frame 51 −2.24 0.0123547 similar to Dynamin-1(D100) (Dynamin, brain) (B-dyn −2.24 0.0123547 similar to Dynamin-1(D100) (Dynamin, brain) (B-dyn −2.24 0.0123547 AE binding protein 1−2.25 0.000105628 golgi autoantigen, golgin subfamily a-like pseudogene−2.26 0.00770626 transmembrane protein 16A −2.27 0.0481085 hypotheticalLOC399844 −2.27 0.000491694 oculomedin −2.27 0.00778869 low densitylipoprotein-related protein 1 (alpha-2- −2.28 4.26E−05 macroglofibronectin leucine rich transmembrane protein 2 −2.28 0.0135122phospholipid transfer protein −2.29 0.00999206 similar to Dynamin-1(D100) (Dynamin, brain) (B-dyn −2.29 0.0122573 SATB homeobox 2 −2.310.039781 similar to TBC1 domain family member 3 (Rab GTPase- −2.320.000870285 tweety homolog 1 (Drosophila) −2.32 0.00450824 CD24 molecule−2.34 0.0340122 chimerin (chimaerin) 1 −2.35 0.0287031 AHA1, activatorof heat shock 90kDa protein ATPase −2.37 0.00979472 homolog bicaudal Chomolog 1 (Drosophila) −2.38 0.0347162 solute carrier family 6(neurotransmitter transporter, ta −2.38 0.00729635 milk fat globule-EGFfactor 8 protein −2.39 0.000987073 WNK lysine deficient protein kinase 1−2.40 1.57E−05 small nucleolar RNA, H −2.41 0.00843141 tweety homolog 3(Drosophila) −2.42 0.000165552 SH3 and PX domains 2B −2.42 0.0244357 WDrepeat and SOCS box-containing 1 −2.44 0.0387851 hypothetical proteinPRO2012 −2.45 0.00756704 golgi autoantigen, golgin subfamily a-likepseudogene −2.46 0.00320764 microfibrillar-associated protein 2 −2.470.0152901 collagen, type XII, alpha 1 −2.47 0.000204664 ST6beta-galactosamide alpha-2,6-sialyltranferase 2 −2.47 0.0216987thioredoxin interacting protein −2.48 0.0135494 latent transforminggrowth factor beta binding protein 2 −2.49 4.08E−05 golgi autoantigen,golgin subfamily a-like pseudogene −2.49 0.00603583 formin bindingprotein 1-like −2.50 0.00290401 maternally expressed 3 −2.52 0.0112259PTK7 protein tyrosine kinase 7 −2.54 0.000116114 ribonuclease P RNAcomponent H1 −2.57 0.0156126 sushi-repeat-containing protein, X-linked 2−2.58 0.0253856 sortilin-related VPS10 domain containing receptor 2−2.58 0.00936311 similar to RIKEN cDNA 1110018M03 −2.59 0.00516476pyridoxal-dependent decarboxylase domain containing 2 −2.60 0.00683647Enah −2.61 0.0077547 asporin −2.62 0.000659873 small Cajal body-specificRNA 17 −2.63 0.0301336 nuclear pore complex interacting protein −2.670.00988632 sushi, von Willebrand factor type A, EGF and pentraxin −2.692.23E−05 dom protein tyrosine phosphatase, receptor type, U −2.690.0270428 collagen, type V, alpha 1 −2.70 0.0166427 nuclear pore complexinteracting protein −2.73 0.0018339 transformer-2 alpha −2.74 0.012256dystrophin related protein 2 −2.79 0.0137557 golgi autoantigen, golginsubfamily a, 8A −2.80 0.0111179 collagen, type VI, alpha 2 −2.810.0149554 transforming growth factor, beta 3 −2.81 0.0287865 trophinin−2.82 0.00298044 hypothetical protein MGC24103 −2.86 0.0346673supervillin −2.87 0.0412717 ADAM metallopeptidase with thrombospondintype 1 −2.90 0.0113968 motif, kinesin family member 26B −2.91 0.00363199nuclear pore complex interacting protein −2.91 0.00160273trichorhinophalangeal syndrome I −2.94 0.00557712 nuclear pore complexinteracting protein −2.96 0.00111223 small nucleolar RNA, C −2.960.00666866 homeobox A2 −2.97 0.0435423 distal-less homeobox 5 −3.000.000640157 dachsous 1 (Drosophila) −3.00 0.00697244 small nucleolarRNA, C −3.06 0.0274043 small nucleolar RNA, C −3.06 0.0274043 nuclearpore complex interacting protein −3.09 0.00583397 small nucleolar RNA, C−3.14 0.0104491 small nucleolar RNA, C −3.14 0.0104491sushi-repeat-containing protein, X-linked −3.16 0.00370941 zinc fingerprotein 521 −3.17 0.00668815 nuclear pore complex interacting protein−3.17 0.00117582 chromosome 9 open reading frame 3 −3.18 0.00410177golgi autoantigen, golgin subfamily a, 8B −3.18 0.0121417 hemicentin 1−3.21 0.0461603 small nucleolar RNA, C −3.24 0.00765575 Kallmannsyndrome 1 sequence −3.25 0.000548703 tenascin C (hexabrachion) −3.268.26E−05 nuclear pore complex interacting protein −3.29 0.00282604nuclear pore complex interacting protein −3.34 0.00263888 homeobox B2−3.36 0.00665994 similar to nuclear pore complex interacting protein−3.41 0.0187322 nuclear pore complex interacting protein −3.460.00354416 cholesterol 25-hydroxylase −3.51 0.0445558 ring fingerprotein 144 −3.52 0.0135334 nuclear pore complex interacting protein−3.55 0.00316496 calbindin 2, 29kDa (calretinin) −3.56 0.0290743 nuclearpore complex interacting protein −3.58 0.00032839 nuclear pore complexinteracting protein −3.60 0.000414309 nuclear pore complex interactingprotein −3.62 0.00283418 nuclear pore complex interacting protein −3.640.000213956 nuclear pore complex interacting protein −3.66 0.000377834KIAA1641 −3.69 0.0191782 UDP-N-acetyl-alpha-D-galactosamine:polypeptideN- −3.72 0.00964109 acetylga nuclear pore complex interacting protein−3.73 0.000352007 leucine rich repeat containing 17 −3.75 0.0263961chromosome 9 open reading frame 3 −3.80 0.0233723 nuclear pore complexinteracting protein −3.82 0.00368967 neurotrimin −3.87 3.78E−06 proteintyrosine phosphatase, receptor type, N −4.02 0.0294569 KIAA1641 −4.020.00659194 — −4.06 0.00488845 KIAA1641 −4.16 0.0170531 integrin, alpha11 −4.16 0.000390317 KIAA1641 −4.27 0.013175 odz, odd Oz −4.280.00172671 transmembrane protein 119 −4.34 0.00801387 plexin domaincontaining 2 −4.44 0.031799 ras homolog gene family, member J −4.590.00197982 homeobox B3 −4.60 0.0354368 similar to Protein KIAA0220 −4.720.0302619 raftlin family member 2 −4.79 0.0260454 WNT1 induciblesignaling pathway protein 1 −5.99 0.000672342 clusterin −6.40 0.0303973serpin peptidase inhibitor, clade F (alpha-2 antiplasmi −6.47 0.00362941sulfatase 2 −6.58 5.88E−05 hephaestin −6.74 0.0123141 junctionaladhesion molecule 2 −7.33 0.0306758 fibronectin type III domaincontaining 1 −7.46 0.0334696 sarcoglycan, delta (35kDadystrophin-associated −7.69 0.000881984 glycoprotei cystatin SN −8.270.0496433 microfibrillar-associated protein 4 −8.67 0.00155578 biglycan−8.70 0.00161284 transmembrane, prostate androgen induced RNA −10.540.000100935 carboxypeptidase E −12.48 0.00738131

Characterization of membrane markers on 2D adherent cells suitable foruse according to the present teachings—the surface antigens expressed by2D adherent cells were examined using monoclonal antibodies. These cellswere stable adhesive cells that were expanded in vitro without the lossof phenotype and without showing signs of karyotypic changes. Flowcytometric analysis of 2D adherent cells' membrane markers showed a highincidence of cells expressing CD105, CD73, CD90 and CD29. Furthermore, ahigh incidence of cells was lacking the expression of CD45, CD34 andCD19, CD11b, CD14 and HLA-DR surface markers (FIG. 2).

Immunomodulation by 2D adherent cells—The immunogenicity of the 2Dadherent cells was investigated next. As shown in FIG. 3, four differentbatches of 2D adherent cells were capable of reducing lymphocyteproliferation, following mitogenic stimuli with Phytohemagglutinin(PHA), as was measured by Thymidine incorporation.

Osteocyte induction—osteocyte differentiation of placenta- or bonemarrow-derived adherent cells in osteogenic induction medium resulted indifferentiation of over 50% of the bone marrow cells, as demonstrated bypositive alizarin red staining (FIG. 4B). On the contrary, none of theplacental derived cells showed any signs of osteogenic differentiation(see FIGS. 4B and 4E and Table 9, below).

TABLE 9 Differentiation summary BM 108 + PLC-11- Plc BM109 3-1 PLC-8-2-1Plc-15-3-4-2 4-3-1 Osteocytes +++ − − − − Adipocytes +++ − − − −

Next, 2D adherent cells from bone marrow or placenta origin werestimulated to differentiate in a modified osteogenic medium comprisingVitamin D and higher concentrations of dexamethasone, a modification ofthe osteogenic differentiation protocol according to previous teachings[Parloni et al. (2008) Stem Cells 26(2): 300-11]. As evident from theresults, over 50% of the bone marrow cells underwent differentiationinto osteocytes, as demonstrated by positive alizarin red staining (seeFIG. 5B). However, none of the placental derived cells showed any signsof osteogenic differentiation (see FIG. 5E and Table 9, hereinabove).

Adipocyte induction—adipocyte differentiation of placenta- or bonemarrow-derived 2D adherent cells in adipocyte induction medium resultedin differentiation of over 50% of the bone marrow derived cells (seeFIG. 4C), as demonstrated by positive oil red staining and by typicalmorphological changes (e.g. accumulation of oil droplets in thecytoplasm). In contrast, none of the placental derived cellsdifferentiated into adipocytes (see FIG. 4F and Table 9, hereinabove).

Next, 2D adherent cells from bone marrow or placenta origin werestimulated to differentiate into adipocytes in a modified mediumcomprising a higher level of Indomethacine, a modification of theadipocyte differentiation protocol according to previous teachings[Parloni et al. (2007), supra]. As evident from the results, over 50% ofthe bone marrow derived cells underwent differentiation into adipocytes(see FIG. 5C), as demonstrated by positive oil red staining and bytypical morphological changes (e.g. accumulation of oil droplets in thecytoplasm). In contrast, none of the placental derived cells exhibitedmorphological changes typical of adipocytes (see FIG. 5F and Table 9,hereinabove).

Example 3 Methods of Generating 3D Adherent Cells Suitable for Use inAccordance with the Present Teachings and the 3D Adherent CellsGenerated Thereby

3D adherent cells (PLX-C) were produced which exhibit differentcharacteristics then the above described 3D adherent cells (PLX, Example1).

Materials and Experimental Procedures

Celligen™ Plug Flow Bioreactor

The production of 3D adherent cells for use in accordance with thepresent invention by Celligen™ (PLX-C cells) was composed of severalmajor steps. The process started by collection of a placenta from aplanned cesarean delivery at term.

Adherent cells were then isolated from whole placentas, grown in tissueculture flasks (2D cultures), harvested and stored in liquid nitrogen as2D-Cell Stock (2DCS), the appropriate amount of 2DCS were thawed, washedand seeded onto carriers in bioreactors for further expansion as3D-culture. After 4-21 days of growth in the bioreactors, cells wereharvested and cryopreserved in gas phase of liquid nitrogen as PLX-C.

Receipt of Human Tissue

All placentas obtained were received from the maternity ward underapproval of the Helsinki Committee of the medical facility. Accordingly,all placenta donors signed an informed consent and Donor Screening andDonor Testing was performed (IPC1). Immediately after taking theplacenta from the donor (during the caesarean procedure), it was placedin a sterile plastic bag and then in a Styrofoam box with ice packs. Theplacenta was delivered and immediately placed in a quarantine area untilreleased to use by Quality Control (QC) and Quality Assurance (QA). Allthe following production steps were performed in a quarantine, cleanroom facility until QC approval of mycoplasma test results arrived andthe cells were release for 2D cell growth.

Recovery and Processing of Adherent Cells

To initiate the process, the whole placenta tissue was cut into piecesunder aseptic conditions under laminar flow hood, washed with Hank'sbuffer solution and incubated for 3 hours at 37° C. with 0.1%Collagenase (1 mg Collagenase/ml tissue). 2D cell medium (2D-Mediumcomprising DMEM supplemented with 10% FBS, fungizone 0.25 μg/ml andgentamycin 50 μg/ml) was added and the digested tissue was roughlyfiltered through a sterile metal strainer, collected in a sterile beakerand centrifuged (10 minutes, 1200 RPM, 4° C.). Using gentle pippeting,suspended cells were then washed with 2D-Medium supplemented withantibiotics, seeded in 80 cm² flasks and incubated at 37° C. in a tissueculture incubator under humidified condition supplemented with 5% CO₂.Following 2-3 days, in which the cells were allowed to adhere to theflask surface, they were washed with PBS and 2D-Medium was added.

Two Dimensional (2D) Cell Growth

Prior to the first passage, growth medium samples of 10% of the totalflask number in quarantine was pooled and taken for mycoplasma testing(IPC2). If cells were found to be negative for Mycoplasma (EZ-PCRMycoplasma kit, Biological Industries, Israel), cells were released fromquarantine. After 1-2 additional passages, cells were transferred to the2D production clean room (2DP). Once in Room 2DP, culture was continuedfor another 3-5 passages (of note, cells were grown in 2D-Mediumsupplemented with antibiotics until passage 3, thereafter cells weregrown in 2D-Medium without antibiotics). IPC-3 sample was taken forimmune phenotype after passage 4. Throughout the process, cultures weregrown in a tissue culture incubator under humidified conditions with 5%CO2 at 37° C. After a total of 6-8 passages (9-16 cell doublings), cellswere collected and cryopreserved as the 2D-Cell Stock (2DCS).

The first passage was usually carried out after 10-15 days. Beginning atpassage 2 and continuing until passage 6-8, cells were passaged when theculture reached 70-80% confluence, usually after 3-5 days (1.5-2doublings). The cells were detached from the flasks using 0.25%trypsin-EDTA (4 minutes at 37° C.) and seeded in a culture density of3±0.2×10³ cells/cm². The size of the tissue culture flasks raised as thepassages proceed. The culturing process started in 80 cm² tissue cultureflask, continued in 175 cm², then in 500 cm² (Triple flask) and finallythe cells were seeded into Cell Factory 10 tray (6320 cm²).

Prior to cryopreservation, at the end of 2DCS growth period, the growthmedium was collected and the sample was prepared to be sent to anapproved GLP laboratory for Mycoplasma test (IPC 4).

Cryopreservation Procedure for 2D-Cell-Stock Product

For 2DCS cryopreservation, 2D-cultured cells were collected underaseptic conditions using 0.25% trypsin-EDTA. The cells were centrifuged(1200 RPM, 10′, 4° C.), counted and re-suspended in 2D-Medium.

For freezing, cell suspensions were diluted 1:1 with 2D-Freezing Mixture(final concentrations was 10% DMSO, 40% FBS and 50% 2D-Medium).Approximately 1.5-2.5×10⁹ cells were manufactured from one placenta. 4ml of the cells were stored at a final concentration of 10×10⁶/ml in 5ml cryopreservation polypropylene vials. The vials were labeled andtransferred to a controlled rate freezer for a graduated temperaturereducing process (1° C./min), after which they were transferred tostorage in gas-phase of a liquid nitrogen freezer located in the ColdStorage Room. This material was referred to as the 2D-Cell Stock (2DCS)batch.

Initiation of the Three Dimensional (3D) Culture Procedures

To begin 3D culture, an appropriate amount (150±30×10⁶) of cells from2DCS were thawed in the 2DP room and washed with 3D-Medium (DMEM with10% FBS and 20 Mm Hepes) to remove DMSO prior to seeding in theprepared-in-advanced bioreactor systems. The content of each 2DCS vialwas pipetted and diluted 1:9 with pre-warmed (37° C.) 3D-Medium. Thecells were centrifuged (1200 RPM, 10′, 4° C.) and re-suspended again in50-100 ml pre-warmed (37° C.) 3D-Medium in a 250 ml sterile bottle. Asample was taken and cells were counted using a Trypan Blue stain inorder to determine cell number and viability. The cell suspension wastransferred under a laminar flow hood into a 0.5 L seeding bottle. Fromthe seeding bottle the cell suspension was transferred via steriletubing to the bioreactor by gravitation.

Production of Adherent Cells in the Celligen Bioreactor (PLX-C)

Bioreactor Description

3D growth phase was performed using an automatic CelliGen Plus® orBIOFLO 310 bioreactor system [(New Brunswick Scientific (NBS)]. Thebioreactor system was used for cultivation of cell culture, in whichconditions were suitable for high cell concentrations. The cultivationprocess was carried out using a bioreactor in a perfusion mode. The labscale bioreactor was constructed of two main systems—the control systemand the bioreactor itself (vessel and accessories). The parameters ofthe process were monitored and controlled by a control console whichincluded connectors for probes, motor and pumps, control loops forDissolved Oxygen (DO), pH, perfusion and agitation (with a motor), agases control system, water circulation and heating system fortemperature control and an operator interface. The controlled processparameters (such as temperature, pH, DO etc.) could be displayed on theoperator interface and monitored by a designated controller.

Cell Culture Growth Procedure in the Bioreactors

As noted in the section hereinabove, 150±30×10⁶ cells from thecryopreserved 2DCS were thawed, washed and seeded in a sterilebioreactor. The bioreactor contained 30-50 gr carriers (FibraCel® disks,NBS), made of Polyester and Polypropylene and 1.5±0.1 L 3D-Medium. Thegrowth medium in the bioreactor was kept at the following conditions:37° C., 70% Dissolved Oxygen (DO) and pH 7.3. Filtered gases (Air, CO₂,N₂ and O₂) were supplied as determined by the control system in order tokeep the DO value at 70% and the pH value at 7.3. For the first 24hours, the medium was agitated at 50 Rounds Per Minutes (RPM) andincreased up to 200 RPM by day 2. For the first 2-3 days, the cells weregrown in a batch mode. Perfusion was initiated when the medium glucoseconcentration decreased below 550 mg/liter. The medium was pumped fromthe feeding container to the bioreactor using sterile silicone tubing.All tubing connections were performed under laminar flow using sterileconnectors. The perfusion was adjusted on a daily basis in order to keepthe glucose concentration constant at approximately 550±50 mg\liter. Asample of the growth medium was taken every 1-2 days for glucose,lactate, glutamine, glutamate and ammonium concentration determination(BioProfile 400 analyzer, Nova Biomedical). The glucose consumption rateand the lactate formation rate of the cell culture enabled to measurecell growth rate. These parameters were used to determine the harvesttime based on accumulated experimental data.

Harvest of the 3D Grown PLX-C Cells from the Bioreactor

The cell harvest process started at the end of the growth phase (4-10days). Two samples of the growth medium were collected. One sample wasprepared to be sent to an approved GLP laboratory for Mycoplasma testingaccording to USP and Eu standards, and the other one was transferred toa controlled rate freezer for a graduated temperature reducing process(1° C./min), after which they were transferred to storage in gas-phaseof a liquid nitrogen freezer located in the Cold Storage Room, in case arepeat Mycoplasma testing was needed. These medium samples wereconsidered as part of the Mycoplasma testing of the final product andthe results were considered as part of the criteria for product release.

The 3D-grown culture was harvested in the Class-100 laminar area in room3DP as follows:

The bioreactor vessel was emptied using gravitation via tubing to awaste container. The vessel was opened, by removing the head plate, andthe carriers were aseptically transferred, using sterile forceps, fromthe basket to the upper basket net. The bioreactor vessel was thenclosed and refilled with 1.5 L pre-warmed PBS (37° C.). The agitationspeed was increased to 150 RPM for 2 minutes. The PBS was drained viatubing by pressure or gravity to the waste bottle. The washing procedurewas repeated twice.

In order to release the cells from the carriers, 1.5 L pre-warmed to 37°C. Trypsin-EDTA (Trypsin 0.25%, EDTA 1 mM) was added to the bioreactorvessel and carriers were agitated for 5 minutes in 150 RPM, 37° C. Cellsuspension was collected to a 5 L sterile container containing 250 mlFBS. Cell suspension was divided to 4 500 ml sterile centrifuge tubesand a Mycoplasma test sample was withdrawn. Closed centrifuge tubes weretransferred through the 3DP active pass-through into the class 10,000filling room (FR1) in which the cells were aseptically filled andcryopreserved as PLX-C.

Cell Cycle Analysis

PLX-C cells obtained by Celligen and PLX cells obtained by Plurix werefixed with 70% EtOH O.N, centrifuged and re-suspended in a PropidiumIodide (PI) solution containing 2 μg/ml PI (Sigma), 0.2 mg/ml Rnase A(Sigma) and 0.1% (v/v) Triton (Sigma) for 30 minutes. Cell cycle wasanalyzed by FACS.

Gene Expression Array (Microarray)

Adherent cells were obtained from human full term placentas and wereexpanded Plurix or by Celligen. Three different batches of cells wereobtained from each of the expansion methods for further examination.

RNA was extracted from the cells (Qiagen-Rneasy micro kit) and appliedto an Affymetrix whole genome expression array. The chip used GeneChip®Human Exon 1.0 ST Array (Affymetrix, Santa Clara, Calif., USA).

FACS Analysis of Membrane Markers

Cells were stained with monoclonal antibodies as previously described.In short, 400,000-600,000 cells were suspended in 0.1 ml flow cytometerbuffer in a 5 ml test tube and incubated for 15 minutes at roomtemperature (RT), in the dark, with each of the following monoclonalantibodies (MAbs): FITC-conjugated anti-human CD29 MAb (eBioscience), PEconjugated anti human CD73 MAb (Becton Dickinson), PE conjugated antihuman CD105 MAb (eBioscience), PE conjugated anti human CD90 MAb (BectonDickinson), FITC-conjugated anti-human CD45 MAb (IQProducts),PE-conjugated anti-human CD19 MAb (IQProducts), PE conjugated anti humanCD14 MAb (IQProducts), FITC conjugated anti human HLA-DR MAb(IQProduct), PE conjugated anti human CD34 MAb (IQProducts), FITCconjugated anti human CD31 MAb (eBioscience), FITC conjugated anti humanKDR MAb (R&D systems), anti human fibroblasts marker (D7-FIB)MAb(ACRIS), FITC-conjugated anti-human CD80 MAb (BD), FITC-conjugatedanti-human CD86 MAb (BD), FITC-conjugated anti-human CD40 MAb (BD),FITC-conjugated anti-human HLA-ABC MAb (BD), Isotype IgG1 FITCconjugated (IQ Products), Isotype IgG1 PE conjugated (IQ Products).

Cells were washed twice with flow cytometer buffer, resuspended in 500μl flow cytometer buffer and analyzed by flow cytometry using FC-500Flow Cytometer (Beckman Coulter). Negative controls were prepared withrelevant isotype fluorescence molecules.

Mixed Lymphocyte Reaction (MLR)

2×10⁵ peripheral blood (PB) derived MNC (from donor A) were stimulatedwith equal amount of irradiated (3000 Rad) PB derived MNCs (from donorB). Increasing amounts of PLX-Cs were added to the cultures. Threereplicates of each group were seeded in 96-well plates. Cells werecultured in RPMI 1640 medium containing 20% FBS. Plates were pulsed with1 μC ³H-thymidine during the last 18 hrs of the 5-day culturing. Cellswere harvested over a fiberglass filter and thymidine uptake wasquantified with scintillation counter.

For CFSE staining, PB-MNC cells were stained for CFSE (Molecular Probes)for proliferation measurement before culturing. Cells were collectedafter 5 days and the intensity of CFSE staining was detected by FlowCytometry.

ELISA

ELISA was carried out as was previously described. In short, MNCs(isolated from peripheral blood) were stimulated with 5 μg/ml ConA(Sigma), 0.5 μg/ml LPS (SIGMA), or 10 μg/ml PHA (SIGMA) in the presenceof PLX-C under humidified 5% CO2 atmosphere at 37° C. Supernatants werecollected and subjected to cytokine analysis using ELISA kits for IFNγ(DIACLONE), TNFα (DIACLONE) and IL-10 (DIACLONE).

Results

The changes in manufacturing with Celligen as compared to Plurixresulted in several major differences (summarized in Table 10, below).

TABLE 10 Comparison between Plurix system (WO/2007/108003) and Celligensystem (teachings of the present invention) 3D adherent cells of theParameter WO/2007/108003 present teachings Improvement Working 280 1500Scale up of the volume (ml) process. Higher production level in thepresent teachings (2-8 population doubling) Weight of  1.4  30 Scale upof the carrier (gr) process in the present teachings. Bed Conic, 50 mlCylinder The present configuration column Packed bed teachings -- Betterflow of medium and nutrients. WO/2007/108003 - Inefficient flow due tonarrow outlet form the conic structure Better homogeneity of mediumflow. Channeling in the plurix Cell 3 × 10⁶ cell/gr 5 × 10⁶ cell/grBetter cell to cell concentration carrier carrier interaction in the atseeding present teachings (cell/gr carrier) Cell 0.015 × 10⁶ cell/ml 0.1× 10⁶ cell/ml Better cell to cell concentration interaction in the atseeding present teachings (cell/ml) Seeding Seeding at low Seeding atthe WO/2007/108003 - procedure medium volume final working Heterogenicfor 24 h followed volume while distribution of the by addition ofagitating cell culture inside medium to final the carrier bed workingvolume Insufficient medium volume in the first 24 h of the run. Leadingto unsuitable working conditions (acidic environment) Production 14-21days 4-10 days Better product phase quality. duration Efficient harvestprocess. Better yield. Lower cost process in the present teachings Modeof Repeated batch - Perfusion Present teachings - operation mediumchange mode - rate Moderate twice a week was adjusted changes of theaccording to conditions the glucose regarding concentration medium (themedium composition was changed throughout the at glucose runconcentration Continuous of 550 ± 50 mg/L) removal of toxic agentsproduced by the cells. In batch mode - lower concentration of essentialnutrients (limiting factors) Less cell debris Harvest Harvesting in 50ml Harvesting Present teachings - procedure tubes inside the Moreefficient Trypsinization 3 bioreactor process cycles TrypsinizationHarvest is carried 1 cycle out in a close system. 1 trypsinizationcycle - better quality of the cells. Agitation medium Cell lift Presentteachings - Circulation impeller Medium is between reservoir flowingthrough container to the the packed bed - column using Better supply ofperistaltic pump nutrients and oxygen to the culture. Homogeneity of themedium Improves other control loops (temp., DO, pH) Temperature Theproduction On-line direct Present teachings - control was carried outcontrol. more accurate inside an Heat transfer measurement of incubator.via water the culture Indirect jacket. temperature. temperature Quickresponse. control (of the Short time to incubator reach set point.chamber). Heat transfer via air interface Temperature Manually. On-linedirect Present teachings - monitoring Indirect water monitoring. Bettertemperature monitoring and monitoring. control of the process. Quickresponse to malfunctions. DO None On-line Present teachings - monitoringmonitoring Better monitoring and control of the process. Quick responseto malfunctions DO control None. On-line direct Present teachings -Introduction of air control of a Better control only specific set of DOlevel. point using Better Air, O₂ and maintenance of a N₂. specifiedworking conditions pH Only visual On-line Present teachings - monitoringmonitoring Control and Better control and control (Phenol red asmonitoring of pH level. part of the Better medium) maintenance of aspecified working conditions Aeration Sparge only OverlayWO/2007/108003 - (sparge as an Aeration by option) sparge creates foamthat might damage the cells.

The changes in the manufacturing process resulted in changes incharacteristics of the obtained adherent cells. These differences aresummarized below.

Cell Cycle Analysis of PLX Manufactured by Plurix Compared to PLX-CManufactured by Celligen

PLX-C cells obtained by Celligen were compared to PLX cells obtained byPlurix in order to examine the distribution of the cells between thedifferent phases of the cell cycle. As is clear from FIGS. 6A-B, PLX-Ccells expanded by Celligen exhibited typical proliferating profile(distribution of cells between the different phases of cell cycle).Specifically, 28% of cells were in S and G2/M phases (FIG. 6A). Theseresults indicated that cells were harvested during proliferation andthat the Celligen bioreactor conditions supported cell growth.

Microarray Comparison Between Plurix and Celligen Obtained Cells

Gene expression arrays enabled to simultaneously monitor genome-wideexpression profiles of adherent cells derived from human full termplacentas expanded by Plurix (PLX) or by Celligen (PLX-C). These resultsenabled to asses the molecular mechanism underlying phenotypic variationbetween cells obtained by these different growth methods (see Table 11,below).

TABLE 11 Gene expression in Plurix cells (WO/2007/108003) compared toCelligen cells (teachings of the present invention) Celligen vs PlurixGene (fold change) p-value(treat) interferon-induced protein withtetratricopeptide 17.52 0.0401812 repeats aldehyde dehydrogenase 1family, member A1 16.76 0.00145807 leukocyte-derived arginineaminopeptidase 13.99 3.88E−06 keratin 27 pseudogene 27 12.25 0.000224998similar to Keratin, type I cytoskeletal 18 (Cytokerati 11.83 0.000304949G protein-coupled receptor, family C, group 5, 10.35 3.39E−05 member Aintegrin, alpha 6 9.84 0.0411667 G protein-coupled receptor 126 8.730.00197635 coagulation factor III (thromboplastin, tissue factor) 7.360.012192 Rho GDP dissociation inhibitor (GDI) beta 7.36 0.00200066signal peptide, CUB domain, EGF-like 3 7.20 0.0255115 interferon-inducedprotein with tetratricopeptide 7.09 0.0139777 repeats dickkopf homolog 1(Xenopus laevis) 7.06 3.06E−07 NAD(P)H dehydrogenase, quinone 1 6.630.000282423 keratin 18 6.46 0.000514523 opioid growth factorreceptor-like 1 5.96 0.00114551 mal, T-cell differentiation protein-like5.95 0.00664216 neurofilament, medium polypeptide 150 kDa 5.86 0.0190611DEP domain containing 1 5.82 0.000370513 cathepsin C 5.72 0.00532262 WAS5.47 0.00178153 serpin peptidase inhibitor, clade B (ovalbumin), 5.440.0190218 member solute carrier family 7, (cationic amino acidtransporte 5.33 0.00688017 interferon-induced protein withtetratricopeptide repea 5.18 0.00357376 NUF2, NDC80 kinetochore complexcomponent, 5.05 0.00276524 homolog (S. cere SHC SH2-domain bindingprotein 1 4.95 0.00430878 thioredoxin reductase 1 4.86 0.000197486 lungcancer metastasis-associated protein 4.85 0.00148024 Rho GTPaseactivating protein 29 4.85 0.0466211 cell division cycle 20 homolog (S.cerevisiae) 4.80 0.00514206 family with sequence similarity 111, memberB 4.63 0.000125819 PDZ binding kinase 4.54 0.00784983 establishment ofcohesion 1 homolog 2 (S. cerevisiae) 4.53 0.000773033 guanylate bindingprotein 4 4.47 0.000215944 lipase A, lysosomal acid, cholesterolesterase 4.42 0.0167385 (Wolman dise kinesin family member 20A 4.390.00582352 KIAA0101 4.28 0.0105909 cyclin-dependent kinase inhibitor 3(CDK2-associated 4.25 0.000732492 dual thymidylate synthetase 4.230.00685584 chromosome 13 open reading frame 3 4.18 0.000548296 aurorakinase A 4.16 0.00632571 nei endonuclease VIII-like 3 (E. coli) 4.140.00115606 centrosomal protein 55 kDa 4.13 0.0021952 oxidized lowdensity lipoprotein (lectin-like) receptor 1 4.11 0.0205198 denticlelesshomolog (Drosophila) 4.05 0.00141153 anillin, actin binding protein 4.010.010923 ribonucleotide reductase M2 polypeptide 3.98 0.00834059 ankyrinrepeat domain 1 (cardiac muscle) 3.93 0.00911953 transcription factor 19(SC1) 3.89 0.00109627 keratin 18 3.89 0.000112551 non-SMC condensin Icomplex, subunit G 3.88 0.00537097 cyclin E2 3.87 0.000203389trypsinogen C 3.86 0.00416276 small nucleolar RNA, C 3.81 0.0334484tight junction protein 2 (zona occludens 2) 3.81 0.00012562 kinesinfamily member 18A 3.78 0.00134108 kinesin family member 2C 3.770.0059888 shugoshin-like 1 (S. pombe) 3.76 0.00101318 polo-like kinase 1(Drosophila) 3.75 0.0140309 thymidine kinase 1, soluble 3.73 0.00124134transcription factor 19 (SC1) 3.73 0.00124327 transcription factor 19(SC1) 3.73 0.00124327 claspin homolog (Xenopus laevis) 3.71 0.00683624GINS complex subunit 1 (Psf1 homolog) 3.69 0.00104515 microsomalglutathione S-transferase 1 3.67 0.041701 arylacetamide deacetylase-like1 3.67 0.000902645 SPC25, NDC80 kinetochore complex component, 3.650.00568662 homolog (S. ce integrin, alpha 4 (antigen CD49D, alpha 4subunit of 3.62 0.0158411 VLA-4 catenin (cadherin-associated protein),alpha-like 1 3.57 7.46E−05 discs, large homolog 7 (Drosophila) 3.560.0317074 v-myb myeloblastosis viral oncogene homolog 3.55 0.0043878(avian)-lik serglycin 3.54 0.0443487 centromere protein N 3.530.000540143 cyclin A2 3.53 0.00965934 heat shock 22 kDa protein 8 3.520.0219583 sema domain, immunoglobulin domain (Ig), short 3.49 0.008548basic doma Rho GTPase activating protein 11A 3.49 0.00834174 Fanconianemia, complementation group I 3.43 0.00464532 BUB1 budding uninhibitedby benzimidazoles 1 3.42 0.0108258 homolog (yeast ovary-specific acidicprotein 3.42 0.00334641 cholinergic receptor, muscarinic 2 3.410.0320078 cell division cycle 2, G1 to S and G2 to M 3.41 0.0017111protein regulator of cytokinesis 1 3.39 0.0325664 minichromosomemaintenance complex component 5 3.38 0.00475504 sperm associated antigen5 3.37 0.00906321 maternal embryonic leucine zipper kinase 3.340.00908391 small nucleolar RNA, C 3.33 0.0298703 carnitinepalmitoyltransferase 1A (liver) 3.33 0.00170894 similar toUbiquitin-conjugating enzyme E2S (Ubiqui 3.33 0.000415822 kinesin familymember 11 3.33 0.00915145 NIMA (never in mitosis gene a)-related kinase7 3.33 0.00159114 ADAM metallopeptidase with thrombospondin type 1 3.320.0102751 motif, transforming, acidic coiled-coil containing protein 33.31 0.0014577 cyclin B1 3.29 0.0103092 MAD2 mitotic arrestdeficient-like 1 (yeast) 3.28 0.00488102 dihydrofolate reductase 3.280.00178879 NIPA-like domain containing 3 3.27 0.00164708 cell divisioncycle associated 2 3.26 0.0122226 apolipoprotein B mRNA editing enzyme,catalytic 3.26 0.00308692 polypep cyclin B2 3.25 0.016544 endonucleasedomain containing 1 3.24 0.000429245 dihydrofolate reductase pseudogene3.23 0.00141306 ATPase, Na+ 3.23 0.000381464 replication factor C(activator 1) 3, 38 kDa 3.23 0.00109668 WD repeat domain 76 3.220.0023531 pleckstrin 2 3.17 0.0304429 Rac GTPase activating protein 13.17 0.00381613 PHD finger protein 19 3.17 0.000177604 deleted inlymphocytic leukemia, 2 3.15 0.0109528 centromere protein I 3.150.0106816 BRCA1 associated RING domain 1 3.14 0.000540414 regulator ofG-protein signalling 4 3.13 0.00781061 STAM binding protein-like 1 3.110.0181743 sulfiredoxin 1 homolog (S. cerevisiae) 3.10 5.14E−05chromosome 15 open reading frame 23 3.08 0.000147331 TTK protein kinase3.08 0.0112171 non-SMC condensin II complex, subunit G2 3.08 0.0130322villin 2 (ezrin) 3.07 0.0131934 stomatin 3.06 0.00387095 proteintyrosine phosphatase-like A domain containing 3.06 0.0419644 serpinpeptidase inhibitor, clade B (ovalbumin), 3.05 0.0030439 member kinesinfamily member 4A 3.05 0.0114203 hypothetical protein DKFZp762E1312 3.050.00726778 ubiquitin-conjugating enzyme E2S 3.04 0.00118205hydroxysteroid dehydrogenase like 2 3.03 3.71E−05 ATPase family, AAAdomain containing 2 3.01 0.00415258 TPX2, microtubule-associated,homolog (Xenopus 3.00 0.0253137 laevis) histone cluster 1, H4d 3.000.030183 kinesin family member 23 2.99 0.00790585 heat shock 70 kDaprotein 2 2.99 0.0215102 origin recognition complex, subunit 1-like(yeast) 2.99 0.00207753 dihydrofolate reductase 2.98 0.00307793hyaluronan-mediated motility receptor (RHAMM) 2.97 0.004678163′-phosphoadenosine 5′-phosphosulfate synthase 2 2.97 1.43E−05glycerol-3-phosphate dehydrogenase 2 (mitochondrial) 2.95 0.00211969nucleolar and spindle associated protein 1 2.95 0.00520875 diaphanoushomolog 3 (Drosophila) 2.95 0.00107709 kinesin family member 14 2.940.00947901 histone cluster 1, H1b 2.93 0.0470898 guanine nucleotidebinding protein (G protein), alpha 2.92 0.00184597 inhi minichromosomemaintenance complex component 8 2.92 0.000841489 cancer susceptibilitycandidate 5 2.92 0.0330594 leukotriene B4 12-hydroxydehydrogenase 2.920.000685452 glutamate-cysteine ligase, modifier subunit 2.91 0.00378868forkhead box M1 2.91 0.0203154 adipose differentiation-related protein2.90 0.000331751 membrane bound O-acyltransferase domain containing 12.90 0.01185 ubiquitin-conjugating enzyme E2T (putative) 2.90 0.00741886cell division cycle associated 3 2.89 0.006289 integrin, alpha 3(antigen CD49C, alpha 3 subunit of 2.88 0.00574148 VLA-3 coagulationfactor XIII, B polypeptide 2.88 0.0294465 RAD51 homolog (RecA homolog,E. coli) (S. cerevisiae) 2.87 0.000854739 ATP-binding cassette,sub-family C (CFTR 2.87 0.00382491 family with sequence similarity 29,member A 2.85 0.00111165 SH2 domain containing 4A 2.84 0.0323646membrane protein, palmitoylated 1, 55 kDa 2.84 0.000396285 CDC28 proteinkinase regulatory subunit 1B 2.84 0.0107391 PSMC3 interacting protein2.84 0.00766442 elastin microfibril interfacer 2 2.84 0.0192072topoisomerase (DNA) II alpha 170 kDa 2.83 0.0321109 transmembraneprotein 106C 2.82 0.000214223 histone cluster 1, H3b 2.80 0.0304598chromosome 18 open reading frame 24 2.80 0.00347442 epidermal growthfactor receptor pathway substrate 8 2.79 0.0194949 high-mobility groupnucleosomal binding domain 2 2.78 0.0030536 SCL 2.78 0.00390288 hectdomain and RLD 4 2.78 0.00679184 ASF1 anti-silencing function 1 homologB (S. cerevisiae) 2.77 0.00543408 thyroid hormone receptor interactor 132.76 0.0118319 cell division cycle associated 8 2.75 0.00619878 kinesinfamily member C1 2.74 0.00821937 high-mobility group nucleosomal bindingdomain 2 2.73 0.00384071 ornithine decarboxylase 1 2.73 0.00144868 v-mybmyeloblastosis viral oncogene homolog 2.71 0.00989416 (avian)-like 2 KITligand 2.70 0.00641955 dual-specificity tyrosine-(Y)-phosphorylation2.70 0.0234606 regulated ki intraflagellar transport 80 homolog(Chlamydomonas) 2.70 0.0247286 transmembrane protein 48 2.69 0.00458248EBNA1 binding protein 2 2.69 0.00296292 ZW10 interactor 2.69 1.88E−05exonuclease 1 2.68 0.00739393 transketolase (Wernicke-Korsakoffsyndrome) 2.68 1.92E−05 somatostatin receptor 1 2.68 0.0144901isocitrate dehydrogenase 3 (NAD+) alpha 2.67 0.00297129 cytoskeletonassociated protein 2 2.67 0.0030499 minichromosome maintenance complexcomponent 4 2.67 0.00342054 inhibitor of DNA binding 1, dominantnegative helix- 2.66 0.036485 loop-hel CDC28 protein kinase regulatorysubunit 1B 2.66 0.0145263 keratin 18 2.66 8.40E−05 CD97 molecule 2.660.00994045 chromosome 6 open reading frame 173 2.64 0.00222408 BTB (POZ)domain containing 3 2.62 0.0166824 deafness, autosomal dominant 5 2.620.00235481 KIAA0286 protein 2.62 0.00130563 Fanconi anemia,complementation group D2 2.61 0.0281405 polo-like kinase 4 (Drosophila)2.60 0.00209633 ribonucleotide reductase M1 polypeptide 2.60 0.000170076malic enzyme 1, NADP(+)-dependent, cytosolic 2.59 0.0435444 non-SMCcondensin I complex, subunit H 2.59 0.0216752 S100 calcium bindingprotein A3 2.58 0.0324073 ubiquitin-conjugating enzyme E2L 3 2.570.00343347 BUB1 budding uninhibited by benzimidazoles 1 2.56 0.0166047homolog beta glycerol kinase 2.55 2.66E−05 TAF9B RNA polymerase II, TATAbox binding 2.54 0.0170365 protein (TBP)-as TAF9B RNA polymerase II,TATA box binding 2.54 0.0170365 protein (TBP)-as histone cluster 1, H2bg2.52 0.000180822 high-mobility group box 2 2.52 0.0196872 NIMA (never inmitosis gene a)-related kinase 2 2.50 0.00289469 proline rich 11 2.500.0357125 myopalladin 2.49 0.0255088 brix domain containing 1 2.490.00471977 cell division cycle associated 5 2.49 0.01021 fucosidase,alpha-L-2, plasma 2.49 0.00540929 cyclin-dependent kinase 2 2.490.00250724 lamin B receptor 2.49 0.000151784 hypoxanthinephosphoribosyltransferase 1 (Lesch- 2.49 0.000634057 Nyhan syndtripartite motif-containing 25 2.47 0.0456344 proteasome (prosome,macropain) subunit, beta type, 9 2.46 0.0202595 (lar proteasome(prosome, macropain) subunit, beta type, 9 2.46 0.0202595 (larproteasome (prosome, macropain) subunit, beta type, 9 2.46 0.0202595(lar sphingomyelin synthase 2 2.46 0.0020701 transmembrane protein 622.45 0.00761064 glucose-6-phosphate dehydrogenase 2.44 0.00278311 PHDfinger protein 1 2.44 0.010191 retinoblastoma-like 1 (p107) 2.440.00319946 KIAA1524 2.43 0.0380688 ST6(alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl- 2.43 0.00830766 1,cofilin 2 (muscle) 2.43 0.0459235 hypothetical protein LOC201725 2.420.000313319 cell division cycle 25 homolog A (S. pombe) 2.42 0.000341692breast cancer 1, early onset 2.41 0.0180553 transaldolase 1 2.410.00199537 mRNA turnover 4 homolog (S. cerevisiae) 2.41 0.00373104glucosaminyl (N-acetyl) transferase 1, core 2 (beta- 2.41 0.01971481,6-N- cysteine rich transmembrane BMP regulator 1 2.41 0.0267286(chordin-like) tissue factor pathway inhibitor (lipoprotein-associated2.40 0.0356227 chromosome 16 open reading frame 59 2.40 0.00185191glycogenin 1 2.39 0.0224317 transmembrane protein 154 2.39 0.0045589tubulointerstitial nephritis antigen-like 1 2.39 0.00510812 CTP synthase2.38 8.80E−05 phenylalanyl-tRNA synthetase, beta subunit 2.380.000245973 geminin, DNA replication inhibitor 2.38 0.00167629 lamin B12.37 0.0477748 SPC24, NDC80 kinetochore complex component, 2.360.00287227 homolog (S. ce glutathione reductase 2.36 0.00353875ribosomal protein L22-like 1 2.36 0.00335381 fumarylacetoacetatehydrolase (fumarylacetoacetase) 2.36 3.88E−05 small nucleolar RNA, C2.35 0.0188991 family with sequence similarity 64, member A 2.350.0019785 epithelial cell transforming sequence 2 oncogene 2.350.000571152 polymerase (DNA directed), epsilon 2 (p59 subunit) 2.340.00479612 glycerol kinase 2.34 3.37E−06 glutathione S-transferase M2(muscle) 2.33 0.0402076 elongation factor, RNA polymerase II, 2 2.330.0130017 thioredoxin 2.33 0.009636 polymerase (DNA directed), alpha 2(70 kD subunit) 2.32 0.0033903 breast cancer 2, early onset 2.320.00586847 CDC45 cell division cycle 45-like (S. cerevisiae) 2.320.00735977 H2A histone family, member Z 2.32 0.0129697 transporter 1,ATP-binding cassette, sub-family B 2.31 0.0164234 (MDR transporter 1,ATP-binding cassette, sub-family B 2.31 0.0164234 (MDR transporter 1,ATP-binding cassette, sub-family B 2.31 0.0164234 (MDR nucleolar complexassociated 3 homolog (S. cerevisiae) 2.30 0.000373346 ATPase, Ca++transporting, plasma membrane 4 2.30 0.023011 minichromosome maintenancecomplex component 7 2.30 0.0457691 TIMELESS interacting protein 2.290.00771062 von Hippel-Lindau binding protein 1 2.28 0.00329061ras-related C3 botulinum toxin substrate 2 (rho family, 2.28 0.0292466sma thymopoietin 2.28 0.0223176 peptidylprolyl isomerase F (cyclophilinF) 2.28 0.00093846 activated leukocyte cell adhesion molecule 2.270.00242163 polycomb group ring finger 5 2.27 0.000294142 Ran GTPaseactivating protein 1 2.27 9.68E−05 replication factor C (activator 1) 4,37 kDa 2.26 0.00164152 tubulin, beta 2C 2.26 0.000346744 minichromosomemaintenance complex component 10 2.26 0.0037925 H2B histone family,member S 2.25 0.000885505 gamma-glutamyl hydrolase (conjugase, 2.250.0195219 folylpolygammaglutamyl transcription termination factor, RNApolymerase II 2.25 0.000393489 polymerase (DNA directed), delta 2,regulatory 2.25 0.0123823 subunit 50k transporter 1, ATP-bindingcassette, sub-family B 2.25 0.00859077 (MDR transporter 1, ATP-bindingcassette, sub-family B 2.25 0.00859077 (MDR transporter 1, ATP-bindingcassette, sub-family B 2.25 0.00859077 (MDR histone cluster 1, H2bf 2.250.0124279 eukaryotic translation initiation factor 1A, X-linked 2.240.00330183 phosphoglucomutase 2 2.24 0.00818204 peroxisomalD3,D2-enoyl-CoA isomerase 2.24 0.00148722 interferon-induced proteinwith tetratricopeptide 2.24 0.0177928 repeats G-2 and S-phase expressed1 2.23 0.0241887 minichromosome maintenance complex component 2 2.230.0021347 family with sequence similarity 72, member A 2.23 0.00143248RMI1, RecQ mediated genome instability 1, homolog 2.23 0.00294705 (S.cerev FLJ20105 protein 2.23 0.0127979 multiple coagulation factordeficiency 2 2.22 0.0116892 phytoceramidase, alkaline 2.22 0.0157729coiled-coil domain containing 68 2.22 0.00227586 dedicator ofcytokinesis 11 2.21 0.00697577 platelet-derived growth factor alphapolypeptide 2.21 0.00176418 N-acylsphingosine amidohydrolase(non-lysosomal 2.20 0.00728536 cerami S-phase kinase-associated protein2 (p45) 2.20 0.00230153 polymerase (RNA) III (DNA directed) polypeptideG 2.20 0.0298794 (32 kD) ADP-ribosylation factor-like 6 interactingprotein 1 2.20 0.00139745 histone cluster 1, H2bh 2.19 0.0377748 originrecognition complex, subunit 5-like (yeast) 2.19 0.049697 CDC28 proteinkinase regulatory subunit 2 2.19 0.0128024 histone cluster 1, H4c 2.190.0112695 hypothetical protein LOC729012 2.19 0.000446087 DEAD(Asp-Glu-Ala-Asp) box polypeptide 39 2.19 0.000340561 chromatin assemblyfactor 1, subunit B (p60) 2.18 0.0119687 MLF1 interacting protein 2.180.0177203 microtubule associated serine 2.18 0.00536974 MHC class Ipolypeptide-related sequence B 2.18 0.0165406 shugoshin-like 2 (S.pombe) 2.18 0.000852557 COP9 constitutive photomorphogenic homolog 2.180.000793512 subunit 6 (Arab methylenetetrahydrofolate dehydrogenase(NADP+ 2.18 0.00119726 dependent) chromosome 6 open reading frame 1672.18 0.0011095 pituitary tumor-transforming 1 2.17 0.0485166ribonuclease H2, subunit A 2.17 0.00669936 X-ray repair complementingdefective repair in 2.16 0.0369865 Chinese ham membrane protein,palmitoylated 5 (MAGUK p55 2.16 0.00211873 subfamily memb karyopherinalpha 2 (RAG cohort 1, importin alpha 1) 2.16 0.000650645 pleckstrinhomology domain containing, family A 2.15 0.0256434 (phosphoi ribosomalprotein L39-like 2.15 0.00429384 karyopherin alpha 2 (RAG cohort 1,importin alpha 1) 2.15 0.000700649 amyloid beta (A4) precursorprotein-binding, family 2.15 0.00201004 B, m minichromosome maintenancecomplex component 3 2.14 0.0018389 histone cluster 1, H2ai 2.140.0129155 chromosome 13 open reading frame 34 2.14 0.000702936 RAD18homolog (S. cerevisiae) 2.14 0.0016685 WD repeat and HMG-box DNA bindingprotein 1 2.13 0.0034833 sulfide quinone reductase-like (yeast) 2.130.0473641 chromosome 16 open reading frame 63 2.12 0.000804179 M-phasephosphoprotein 1 2.12 0.0271814 minichromosome maintenance complexcomponent 6 2.12 0.0161279 homeobox A9 2.11 0.00520942 fibroblast growthfactor 9 (glia-activating factor) 2.10 0.0475844 cell division cycle 25homolog C (S. pombe) 2.10 0.0169914 chromosome 9 open reading frame 642.10 0.0265979 U2AF homology motif (UHM) kinase 1 2.09 0.0255167replication factor C (activator 1) 2, 40 kDa 2.09 0.00768959hypothetical protein LOC440894 2.09 0.0103358 small nuclearribonucleoprotein D1 polypeptide 2.09 0.0334665 16 kDa CSE1 chromosomesegregation 1-like (yeast) 2.09 0.0013662 phosphatidylinositol glycananchor biosynthesis, class W 2.09 0.0151967 centromere protein O 2.090.00397056 family with sequence similarity 20, member B 2.09 0.00460031hypothetical protein FLJ40869 2.09 0.00444509 guanine nucleotide bindingprotein (G protein), 2.08 0.00140559 gamma 11 calcyclin binding protein2.08 0.00524566 ATP-binding cassette, sub-family E (OABP), member 1 2.080.00454751 CD44 molecule (Indian blood group) 2.08 0.000651436 exosomecomponent 8 2.08 0.00132017 family with sequence similarity 102, memberB 2.08 0.025743 histone cluster 2, H3d 2.07 0.0102932 family withsequence similarity 33, member A 2.07 0.000318673 Fanconi anemia,complementation group B 2.07 0.000255109 kinesin family member 22 2.070.0192406 histone cluster 1, H2ai 2.07 0.0161621 vaccinia related kinase1 2.06 0.0233182 integrator complex subunit 7 2.06 0.000841371 flapstructure-specific endonuclease 1 2.06 0.006882 hypothetical proteinFLJ25416 2.06 0.000177531 ecotropic viral integration site 2B 2.060.0171408 retinitis pigmentosa 2 (X-linked recessive) 2.05 0.0264185centromere protein L 2.05 0.000880856 cofactor required for Sp1transcriptional activation, 2.04 0.00141809 subu chromosome 20 openreading frame 121 2.04 0.0146323 family with sequence similarity 72,member A 2.04 0.00162905 family with sequence similarity 72, member A2.04 0.00165234 eukaryotic translation initiation factor 1A, X-linked2.04 0.00520549 elongation factor, RNA polymerase II, 2 2.03 0.0458007ATPase, Na+ 2.03 0.0189108 histone cluster 1, H3a 2.03 0.0244273 brixdomain containing 1 2.03 0.00981178 sushi domain containing 1 2.030.0258164 ectonucleoside triphosphate diphosphohydrolase 6 2.030.00423628 (putativ fructosamine 3 kinase 2.03 0.00470972 Bloom syndrome2.02 0.0209259 tubulin, alpha 1c 2.01 0.00862586 E2F transcriptionfactor 2 2.01 0.0496479 exosome component 2 2.01 0.00649147 kinesinfamily member 22 2.01 0.0242075 LTV1 homolog (S. cerevisiae) 2.010.00812652 dihydrolipoamide S-acetyltransferase (E2 component 2.010.00179011 of pyruv v-ral simian leukemia viral oncogene homolog B (ras2.01 0.012225 related ring finger and WD repeat domain 3 2.01 0.0013797annexin A1 2.01 0.0173578 elaC homolog 2 (E. coli) 2.00 0.00266504aldehyde dehydrogenase 9 family, member A1 2.00 0.00911609 tubulin,alpha 4a 2.00 0.0435427 nuclear pore complex interacting protein −2.000.00111223 oculomedin −2.01 0.00778869 similar to PI-3-kinase-relatedkinase SMG-1 −2.01 0.0356628 golgi autoantigen, golgin subfamily a-likepseudogene −2.01 0.00770626 spectrin repeat containing, nuclear envelope1 −2.01 0.00438469 nuclear pore complex interacting protein −2.010.00117582 sushi, nidogen and EGF-like domains 1 −2.01 0.00161129integrin, alpha V (vitronectin receptor, alpha −2.02 0.00252702polypeptide cyclin-dependent kinase inhibitor 2B (p15, inhibits −2.040.0150268 CDK4) lysyl oxidase-like 4 −2.04 0.0120148 nuclear porecomplex interacting protein −2.04 0.000213956 calcium −2.04 0.00657494calsyntenin 3 −2.04 0.00300887 cell adhesion molecule 1 −2.05 0.0261129solute carrier family 22 (organic cation transporter), −2.05 0.0137275RUN and FYVE domain containing 3 −2.05 0.00387265 glucosidase, alpha;acid (Pompe disease, glycogen −2.05 0.000418401 storage di nuclear porecomplex interacting protein −2.05 0.00988632 proline-rich nuclearreceptor coactivator 1 −2.06 0.0039587 membrane metallo-endopeptidase−2.06 0.0152684 PHD finger protein 21A −2.06 0.00980401 RhoGTPase-activating protein −2.06 0.00705186 homeobox B6 −2.06 0.00301714nuclear pore complex interacting protein −2.07 0.00032839 phospholipaseA2 receptor 1, 180 kDa −2.07 0.00069343 nuclear pore complex interactingprotein −2.08 0.000352007 slit homolog 3 (Drosophila) −2.08 0.02844nuclear pore complex interacting protein −2.09 0.000414309cyclin-dependent kinase 6 −2.09 0.0456892 dynamin 1 −2.09 0.00139674jumonji, AT rich interactive domain 1B −2.09 0.00861002 calcium bindingand coiled-coil domain 1 −2.09 0.00370041 insulin-like growth factor 1receptor −2.09 0.00114467 nuclear pore complex interacting protein −2.100.000377834 CD82 molecule −2.10 0.0175517 bromodomain adjacent to zincfinger domain, 2B −2.10 9.88E−05 — −2.10 0.00666187 synaptotagmin XI−2.11 0.0129428 KIAA1546 −2.11 0.000255634 jun B proto-oncogene −2.120.0120169 CXXC finger 6 −2.12 0.0277527 nuclear pore complex interactingprotein −2.14 0.00282604 Cdon homolog (mouse) −2.15 0.0350357 B-cell CLL−2.15 0.00343507 nuclear pore complex interacting protein −2.150.00263888 v-abl Abelson murine leukemia viral oncogene −2.16 0.0136688homolog 1 nuclear pore complex interacting protein −2.16 0.00583397 FATtumor suppressor homolog 1 (Drosophila) −2.18 0.0158766 transformer-2alpha −2.18 0.012256 chimerin (chimaerin) 1 −2.18 0.0287031 milk fatglobule-EGF factor 8 protein −2.18 0.000987073 vitamin D(1,25-dihydroxyvitamin D3) receptor −2.19 0.000192208 neuroblastoma,suppression of tumorigenicity 1 −2.20 0.00090639 jumonji domaincontaining 1A −2.20 0.0188513 WNK lysine deficient protein kinase 1−2.21 1.57E−05 protocadherin beta 14 −2.21 0.0103892 cortactin bindingprotein 2 −2.21 2.28E−05 WW domain containing transcription regulator 1−2.22 0.0379899 cyclin L1 −2.22 0.00831474 nuclear factor of activatedT-cells, cytoplasmic, −2.22 0.00786451 calcine pellino homolog 1(Drosophila) −2.23 0.00939357 golgi autoantigen, golgin subfamily a-likepseudogene −2.24 0.00603583 chromosome 7 open reading frame 10 −2.260.00738442 golgi autoantigen, golgin subfamily a-like pseudogene −2.270.00320764 small Cajal body-specific RNA 17 −2.27 0.0301336 latenttransforming growth factor beta binding protein 2 −2.29 4.08E−05 golgiautoantigen, golgin subfamily a, 8A −2.29 0.0111179 inhibin, beta A(activin A, activin AB alpha −2.29 0.00877271 polypeptide) solutecarrier family 41, member 2 −2.30 0.00453672 forkhead box P1 −2.300.0463138 matrix metallopeptidase 14 (membrane-inserted) −2.31 1.93E−05transcription factor 4 −2.31 0.0367869 jun oncogene −2.32 7.21E−05neuroepithelial cell transforming gene 1 −2.33 0.0109689 asporin −2.330.000659873 v-fos FBJ murine osteosarcoma viral oncogene −2.35 0.0138624homolog ephrin-B2 −2.36 0.00611474 WD repeat and SOCS box-containing 1−2.36 0.0387851 similar to dJ402H5.2 (novel protein similar to wo −2.360.00621503 PX domain containing serine −2.38 0.000927628 collagen, typeVII, alpha 1 (epidermolysis bullosa, −2.38 0.00109233 dystr AE bindingprotein 1 −2.39 0.000105628 peroxidasin homolog (Drosophila) −2.400.00219049 calcium channel, voltage-dependent, L type, alpha 1C −2.410.0189661 sub Prader-Willi syndrome chromosome region 1 −2.45 0.0415526midline 1 (Opitz −2.45 0.00130803 nuclear pore complex interactingprotein −2.45 0.00354416 chromosome 1 open reading frame 54 −2.470.0186089 transmembrane protein 16A −2.48 0.0481085 basichelix-loop-helix domain containing, class B, 2 −2.49 0.00270257 nuclearpore complex interacting protein −2.50 0.00316496 runt-relatedtranscription factor 1 (acute myeloid −2.50 0.000607387 leukemi zincfinger protein 292 −2.50 0.029832 fibronectin leucine rich transmembraneprotein 2 −2.51 0.0135122 nuclear pore complex interacting protein −2.510.00283418 potassium voltage-gated channel, subfamily G, −2.54 0.0244306member 1 interleukin 19 −2.54 0.0310328 transforming growth factor, beta3 −2.54 0.0287865 dihydropyrimidinase-like 3 −2.55 0.0165203 golgiautoantigen, golgin subfamily a, 8B −2.56 0.0121417 hypothetical proteinPRO2012 −2.57 0.00756704 SATB homeobox 2 −2.57 0.039781 t-complex 11(mouse)-like 2 −2.57 0.0324227 ring finger protein 122 −2.57 0.0236621chromosome 8 open reading frame 57 −2.59 0.00261522 ADAMmetallopeptidase with thrombospondin type 1 −2.60 0.0113968 motif,sushi, von Willebrand factor type A, EGF and −2.63 2.23E−05 pentraxindom ST6 beta-galactosamide alpha-2,6-sialyltranferase 2 −2.64 0.0216987sortilin-related VPS10 domain containing receptor 2 −2.65 0.00936311protocadherin beta 9 −2.66 0.0285124 chromosome 5 open reading frame 13−2.67 0.00410172 Enah −2.68 0.0077547 pyridoxal-dependent decarboxylasedomain containing 2 −2.69 0.00683647 similar to nuclear pore complexinteracting protein −2.70 0.0187322 nuclear pore complex interactingprotein −2.70 0.00368967 transmembrane protein 119 −2.70 0.00801387chromosome 14 open reading frame 37 −2.70 0.0182453sushi-repeat-containing protein, X-linked 2 −2.71 0.0253856 PDZ domaincontaining RING finger 3 −2.71 0.00931014 collagen, type XII, alpha 1−2.72 0.000204664 matrix-remodelling associated 5 −2.72 0.000317637collagen, type V, alpha 1 −2.72 0.0166427 dystrophin related protein 2−2.72 0.0137557 ATP-binding cassette, sub-family A (ABC1), member 1−2.73 0.00131361 trophinin −2.77 0.00298044 cornichon homolog 3(Drosophila) −2.78 0.0261738 formin binding protein 1-like −2.780.00290401 brain and acute leukemia, cytoplasmic −2.78 0.0476919 proteintyrosine phosphatase, receptor type, U −2.80 0.0270428 hypotheticalprotein MGC24103 −2.82 0.0346673 interferon induced with helicase Cdomain 1 −2.83 0.0024839 phospholipid transfer protein −2.84 0.00999206immediate early response 3 −2.87 0.0152127 immediate early response 3−2.87 0.0152127 ADAM metallopeptidase domain 12 (meltrin alpha) −2.870.000870288 synaptic vesicle glycoprotein 2A −2.88 0.00704212 chromosome9 open reading frame 3 −2.88 0.00410177 thioredoxin interacting protein−2.90 0.0135494 early growth response 1 −2.93 0.000425035 smallnucleolar RNA, C −2.94 0.00666866 small nucleolar RNA, C −2.950.00765575 immediate early response 3 −2.99 0.0167309 low densitylipoprotein-related protein 1 (alpha-2- −2.99 4.26E−05 macroglo bicaudalC homolog 1 (Drosophila) −2.99 0.0347162 homeobox B2 −3.03 0.00665994small nucleolar RNA, C −3.10 0.0274043 small nucleolar RNA, C −3.100.0274043 matrix metallopeptidase 2 (gelatinase A, 72 kDa −3.13 5.59E−05gelatinase, KIAA1641 −3.14 0.00659194 collagen, type VI, alpha 3 −3.142.09E−06 homeobox A2 −3.15 0.0435423 SH3 and PX domains 2B −3.150.0244357 collagen, type VI, alpha 2 −3.16 0.0149554 chromosome 9 openreading frame 3 −3.21 0.0233723 small nucleolar RNA, C −3.24 0.0104491small nucleolar RNA, C −3.24 0.0104491 — −3.27 0.00488845UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- −3.35 0.00964109acetylga cholesterol 25-hydroxylase −3.38 0.0445558 KIAA1641 −3.400.013175 ring finger protein 144 −3.40 0.0135334 versican −3.41 0.023885angiopoietin-like 2 −3.42 0.0245161 KIAA1641 −3.44 0.0170531 FBJ murineosteosarcoma viral oncogene homolog B −3.54 0.00025573 similar to RIKENcDNA 1110018M03 −3.59 0.00516476 early growth response 2 (Krox-20homolog, −3.62 0.00821813 Drosophila) dachsous 1 (Drosophila) −3.630.00697244 kinesin family member 26B −3.64 0.00363199 distal-lesshomeobox 5 −3.66 0.000640157 similar to Protein KIAA0220 −3.69 0.0302619insulin-like growth factor 1 receptor −3.71 3.42E−05 protein tyrosinephosphatase, receptor type, N −3.77 0.0294569 KIAA1641 −3.85 0.0191782sushi-repeat-containing protein, X-linked −3.85 0.00370941microfibrillar-associated protein 2 −3.91 0.0152901 complement component1, s subcomponent −3.97 0.0395863 CD24 molecule −3.99 0.0340122 homeoboxB3 −4.02 0.0354368 trichorhinophalangeal syndrome I −4.02 0.00557712Kallmann syndrome 1 sequence −4.04 0.000548703 leucine rich repeatcontaining 17 −4.09 0.0263961 plexin domain containing 2 −4.32 0.031799PTK7 protein tyrosine kinase 7 −4.42 0.000116114 supervillin −4.430.0412717 zinc finger protein 521 −4.58 0.00668815 calbindin 2, 29 kDa(calretinin) −4.77 0.0290743 ras homolog gene family, member J −4.790.00197982 integrin, alpha 11 −4.80 0.000390317 odz, odd Oz −5.050.00172671 F-box protein 32 −5.52 0.0212957 raftlin family member 2−5.72 0.0260454 clusterin −5.74 0.0303973 neurotrimin −5.79 3.78E−06WNT1 inducible signaling pathway protein 1 −5.86 0.000672342insulin-like growth factor binding protein 5 −6.34 0.011614 sulfatase 2−6.34 5.88E−05 microfibrillar-associated protein 4 −6.93 0.00155578junctional adhesion molecule 2 −7.07 0.0306758 fibronectin type IIIdomain containing 1 −7.29 0.0334696 sarcoglycan, delta (35 kDadystrophin-associated −7.37 0.000881984 glycoprotei hephaestin −7.530.0123141 serpin peptidase inhibitor, clade F (alpha-2 antiplasmi −7.660.00362941 cystatin SN −7.96 0.0496433 hemicentin 1 −8.18 0.0461603tenascin C (hexabrachion) −8.32 8.26E−05 biglycan −8.62 0.00161284transmembrane, prostate androgen induced RNA −11.20 0.000100935carboxypeptidase E −11.22 0.00738131

Expression of Cellular Markers on PLX-C Cells

The surface antigens expressed by PLX-C were examined using monoclonalantibodies. Results indicated that PLX-C cells were characterized by thepositive markers: CD73, CD29 and CD105 and the negative markers: CD34,CD45, CD19, CD14 and HLA-DR (data not shown). The immune phenotype testspecifications were set as: ≧90% for all positive markers and ≦3% forall negative markers.

Furthermore, as shown in FIGS. 7A-B, PLX-C cultures did not expressendothelial markers as shown by negative staining for the twoendothelial markers CD31 and KDR. However, PLX-C expression of afibroblast-typical marker was evident (expression of D7-fib, FIG. 7C).

Immunogenecity and Immunomodulatory Properties of PLX-C Cells

As PLX-C is comprised of adherent cells derived from placenta, it isexpected to express HLA type I, which is expressed by all cells of thebody and is known to induce an alloreactive immune response. HLA type IIand other co-stimulatory molecules are typically expressed only on thesurface of Antigen Presenting Cells (APCs).

In order to examine the immunogenicity of the obtained PLX-C cells, theexpression of co-stimulatory molecules on the surface of these cellmembranes were performed. FACS analysis demonstrated the absence ofCD80, CD86 and CD40 on the PLX-C cell membranes (FIGS. 8A-C). Moreover,PLX-C expressed low levels HLA class I as detected by staining for HLAA/B/C (FIG. 8D). The expression of stimulatory and co-stimulatorymolecules was similar to bone marrow (BM) derived MSCs (as shown inFIGS. 8A-D).

To further investigate the immunogenecity as well as theimmunomodulation properties of PLX-C cells, Mix Lymphocyte Reaction(MLR) tests were performed. As shown in FIGS. 9A-B, PLX-C cells bothescape allorecognition and reduce T cell response, as measured byThymidine incorporation. Furthermore, the reduction in lymphocytesproliferation (evaluated by CPM measurement) was higher as the number ofPLX-C cells increased (in a dose dependent manner). PLX-C also reducedlymphocyte proliferation following mitogenic stimuli, such as ConcavalinA (Con A, FIG. 9B) and Phytohemagglutinin (PHA), and non-specificstimulation by anti-CD3, anti-CD28 (data not shown).

In order to investigate the mechanism of action by which PLX-Cimmunomodulate lymphocyte proliferation, and to see if this action ismediated via cell to cell interaction or cytokines secretion, PB derivedMononuclear cells (MNCs) were stimulated by PHA using the transwellmethod (which prevents cell to cell contact but enables the diffusion ofcytokines between the two compartments). Results showed that theinhibition of proliferation maintained even when cell to cell contactwas inhibited (data not shown).

Cytokines Secretion

As depicted hereinabove, PLX-C reduce the proliferation rate oflymphocytes, probably through soluble factors. Further investigation ofthe cytokines secreted by lymphocytes in response to PLX-C was performedto elucidate the mechanism of action of PLX-C. As depicted in FIGS.10A-B, culturing of mononuclear cells with PLX-C slightly reduced thesecretion of the pro-inflammatory cytokine INFγ and dramatically reducedthe secretion of TNFα (even in the presence of low amounts of PLX-C). Inaddition, following lipopolysaccharide (LPS) stimulation, PB derivedMNCs secretion of IL-10 increased in the presence of PLX-C, while thesecretion level of TNFα decreased, in a dose dependent manner (FIG.10C).

It will be appreciated that the PLX-C cells of the present teachingswere also capable of homing to ischemic tissues following intramuscularor intravenous injection into mice (data not shown).

Example 4 The Anti-Inflammatory Effect of PLX-C Cells in an In VivoMurine Model of Acute Colitis

Materials and Experimental Procedures

TNBS Model of Intestinal Inflammation

Colitis was induced in susceptible strains of rodents by intrarectalinstillation of the haptenating substance TNBS (Trinitrobenzyl sulphonicacid) in ethanol. The use of TNBS in Ethanol was based on previousreports that ethanol is required to break the mucosal barrier whereasTNBS haptenizes colonic autologous or microbiota proteins rendering themimmunogenic to the host immune system [Wirtz et al., Nature Protocols(2007) 2(3): 541-546].

Briefly, for colitis induction, mice were anesthetized for 90-120minutes and received an intrarectal administration of TNBS (40 μl, 150mg/kg) dissolved in a 1:1 mixture of 0.9% NaCl in 100% ethanol. Controlmice received a 1:1 mixture of 0.9% NaCl in 100% ethanol or a salinesolution using the same technique.

Mice were sacrificed 5 days post TNBS administration to assess theanti-inflammatory effect of the therapeutic cells (PLX-C cells) of thepresent invention. PLX-C administration was evaluated by intravenous(iv) administration or by intraperitoneal (ip) injection of the cells 1day post colitis induction.

Animals

C57b16 mice were used in these experiments. A total of 90 mice were usedwhich were divided into 9 groups as follows:

1) 10 control mice (received no treatment)2) 10 control mice+PLX-C-I batch 1 by ip (2*10⁶ cells)3) 10 control mice+PLX-C-I batch1 by iv (1*10⁶ cells)4) 10 TNBS mice (colitis model mice)5) 10 TNBS mice+5-aminosalicylic acid (5-ASA)6) 10 TNBS mice+PLX-C-I batch 1 by iv (1*10⁶ cells)7) 10 TNBS mice+PLX-C-I batch 1 by ip (2*10⁶ cells)8) 10 TNBS mice+PLX-C-I batch 2 by iv (1*10⁶ cells)9) 10 TNBS mice+PLX-C-I batch 2 by ip (2*10⁶ cells)

Production of the 2D Placenta Derived Adherent Cells

As depicted in detail in Example 2, hereinabove.

Production of the 3D Placenta Derived Adherent Cells (PLX-C Cells)

As depicted in detail in Example 3, hereinabove.

Vehicle

PlasmaLyte containing 5% Albumin was used as a vehicle control.

Tests and Evaluation

Macroscopic and histological assessments of colitis were performed oncolon specimens collected from the different mice experimental groups 5days post TNBS administration. Macroscopic and histological assessmentswere conducted blindly by two investigators.

Macroscopical Analysis

The colon of each mouse was examined under a dissecting microscope(magnification, ×5) to evaluate the macroscopic lesions according to theWallace criteria. The Wallace score rated the macroscopic lesions on ascale from 0 to 10 based on features reflecting inflammation, such ashyperemia, thickening of the bowel, and extent of ulceration.

TABLE 12 Wallace score Score Criteria of macroscopic evaluation 0 NoInflammation 1 Hyperemia without ulcerations 2 Hyperemia with thickeningof the mucosa without ulcerations 3 1 ulceration without thickening ofthe colonic wall 4 2 or more of ulcerative or inflammatory sites 5 2 ormore of ulcerative or inflammatory sites with an extent of more than 1cm 6 1 ulcerative or inflammatory site of more than 2 cm 7 1 ulcerativeor inflammatory site of more than 3 cm 8 1 ulcerative or inflammatorysite of more than 4 cm 9 1 ulcerative or inflammatory site of more than5 cm 10 1 ulcerative or inflammatory site of more than 6 cm

Histological Analysis

A colon specimen located precisely 2 cm above the anal canal was usedfor the histological evaluation according to the Ameho criteria. Thisgrading (on a scale from 0 to 6) took into account the degree ofinflammation infiltrate, the presence of erosion, ulceration, ornecrosis, and the depth and surface extension of lesions.

TABLE 13 Ameho criteria Score Criteria of histological evaluation 0 Noalterations 1 Middle mucosal and/or sub-mucosal inflammatory infiltrateswith oedema. Few mucosal erosions. Integrity of the muscularis mucosae.2 Same criteria as score 1 but more than 50% of the section 3 Largeinflammatory infiltrate with ulceration area trough all the colonic 4Same criteria as score 3 but more than 50% of the section 5 Wideulcerations with cellular necrosis 6 Wide ulcerations with cellularnecrosis but more than 50% of the section

Molecular Analysis of Colitis

Quantification of mRNA Expression of IL-1 Beta

Total RNA was isolated from whole mice colonic tissues using Rneasy kit(Macherey Nagel, Hoerdt, France) according to the manufacturer'sinstructions. RNA quantification was performed using spectrophotometry.After treatment at 37° C. for 30 minutes with 20-50 units of RNase-freeDNase I (Roche Diagnostics Corporation, Indianapolis, Ind., USA),oligo-dT primers (Roche Diagnostics Corporation, Indianapolis, USA) wereused to synthesize single-stranded cDNA. mRNAs were quantified usingSYBR green Master Mix (Applera, Courtaboeuf, France) with mouse specificoligonucleotides for IL-1β: S: 5′-gATCCACACTCTCCAgCTgCA-3′ (SEQ IDNO: 1) and AS: 5′-CAACCAACAAgTgATATTCTCCATg-3′ (SEQ ID NO: 2) in aGeneAmp Abiprism 7000 (Applera, Courtaboeuf, France). Each assay wascalibrated and no-template controls were included. Each sample was runin triplicate. SYBR green dye intensity was analyzed using the Abiprism7000 SDS software (Applera, Courtaboeuf, France). All results werenormalized to the unaffected housekeeping gene β-actin (oligonucleotidesfor β-actin: S: 5′-gggTCAgAAggATTCCTATg-3′ SEQ ID NO: 3; AS: 5′ggTCTCAAACATgATCTggg-3′ SEQ ID NO: 4).

Results

As described in detail hereinabove, mice were sacrificed on day 5following induction of colitis by intra-rectal injection of TNBS on day0 and adherent cell administration on day 1. Mice were administered witheither 2D adherent cells (hereinafter batch 1) or PLX-C adherent cells(hereinafter batch 2) obtained from placenta 1 or placenta 2,respectively. After the mice were sacrificed, macroscopic andmicroscopic evaluations of the colon were performed.

As demonstrated in FIG. 11, mice treated by an iv injection of 2D andPLX-C cells (batch 1 or 2, respectively) exhibited a major improvementin the inflammatory condition of the colon tissue as represented by theWallace score. This anti-inflammatory effect was as efficient as the5-ASA gold standard treatment). It will be appreciated that ipadministration of 2D adherent cells (batch 1) also resulted in asatisfactory improvement in the Wallace score of the colitis model mice.

Microscopic evaluation of the colon revealed that administration ofPLX-C cells (batch 2) by either ip or iv routes significantly reducedcolonic inflammation at the histological level compared to TNBS mice (asrepresented by the Ameho score, FIG. 12). A considerable improvement wasalso observed for TNBS mice who received 2D cells (batch 1) by ivadministration (FIG. 12). The improvement in these treatment groups wassignificantly better compared to the 5-ASA treated TNBS group.

Furthermore, total RNA was isolated from colonic tissues and IL-1βexpression levels were evaluated by RT-PCR (as described in detailhereinabove). As evident from the results (FIG. 13), administration ofPLX-C cells (batch 2) by iv significantly reduced the expression levelof IL-1β in colonic tissues. It will be appreciated that although IL-1βRNA expression levels were not significantly reduced by ipadministration (2D or 3D adherent cells, batches 1 and 2, respectively)and iv administration (2D adherent cells, batch 1), administration ofboth PLX-C and 2D cells still resulted in significant reduction ininflammation based on macroscopic and microscopic evaluations in coliticmice.

Taken together these results demonstrated that administration of theplacental adherent cells of the present invention (2D and 3D PLX-Ccells) lead to a significant improvement in colon inflammation in amouse model of acute colitis.

Example 5 The Anti-Inflammatory Effect of PLX-C Cells in an In-Vivo RatModel of Acute Colitis

Materials and Experimental Procedures

TNBS Model of Intestinal Inflammation

Rats were rendered colitic by intracolonic administration of 22 mg ofTNBS dissolved in 1:1 mixture of EtOH and water. 24 hours post colitisinduction rats were administered according to the study treatment.

The rats used in this study were divided into three study groups (asdepicted in detail below). Eleven days following colitis induction, allrats were sacrificed and colonic damage was evaluated bothmicroscopically and macroscopically.

Administration of TNBS was designated as day 0, PLX-C cells wereadministrated on day 1 and rats were sacrificed on day 11.

Animals

12 Female Lewis rats (100-120 g) were used in these experiments. A totalof 12 rats were used which were divided into 4 groups as follows:

1) 4 rats were administered ip with 5×10⁶ PLX-C-I cells2) 4 rats were administered iv with 5×10⁶ PLX-C-I cells3) 2 rats received PlasmaLyte by ip (control group)4) 2 rats received PlasmaLyte by iv (control group)

Production of the Placenta Derived Adherent Cells (PLX-C Cells)

Cells were produced as depicted in detail in Example 3, hereinabove.

Macroscopic Assessment of Colonic Damage

Macroscopic assessment of colonic damage was evaluated according to thefollowing criteria:

0—No damage

1—Hyperemia but no ulcers

2—Fibrosis but no ulcers

3—Ulceration/necrosis less than 1 cm

4—Ulceration/necrosis less than 2 cm

5—Ulceration/necrosis more than 2 cm

Microscopic (Histological) Assessment of Colonic Damage

Microscopic assessment of colonic damage was evaluated according to allof the following criteria (A+B+C+D):

A. Extent of ulceration:

0—No ulcer

1-2—Small ulcers (less than 3 mm)

3-5—Large ulcers (more than 3 mm)

B. Submucosal infiltration:

0—None

1—Mild

2-3—Moderate

4-5—Severe

C. Crypt abscesses:

0— None

1-2—Rare

3-5—Diffuse

D. Wall thickness (m)

0—less than 470

1—less than 600

2—less than 700

3—less than 800

4—less than 900

5—more than 900

Results

As evident from FIG. 14, administration of the 3D adherent cells of thepresent invention (PLX-C cells) lead to a significant improvement in themicroscopic scores (histological assessment) of acute colitis in rats.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by into thespecification, to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting.

1-21. (canceled)
 22. A dispenser device comprising: a dispenser forintramuscular or intravascular administration of cells, wherein thedispenser contains a composition comprising a population of adherentcells derived from placenta, wherein said adherent cells do not exhibitosteogenic differentiation as assessed by Alizarin Red staining, whensubjected to conditions that induce bone marrow cells to undergoosteogenic differentiation.
 23. The dispenser device of claim 22,wherein said adherent cells express one or more of CD73, CD90, CD29, orCD105.
 24. The dispenser device of claim 22, wherein said adherent cellsdo not express CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD14, CD19, or CD79.25. The dispenser device of claim 22, wherein said adherent cellssuppress an immune reaction by suppressing T cell activity.
 26. Thedispenser device of claim 22, wherein said adherent cells are obtainedfrom a three-dimensional (3D) culture.
 27. The dispenser device of claim26, wherein said three-dimensional (3D) culture comprises a 3Dbioreactor.
 28. The dispenser device of claim 26, wherein culturing ofsaid adherent cells in said 3D culture comprises perfusion of theadherent cells with a continuous flow of a culture medium.
 29. Thedispenser device of claim 26, wherein culturing of said adherent cellsin said 3D culture occurs for at least 3 days.
 30. The dispenser deviceof claim 26, wherein culturing of said adherent cells in said 3D cultureis performed until at least 10% of said adherent cells areproliferating.
 31. The dispenser device of claim 26, wherein saidadherent cells are cultured from the placenta under 2 dimensional (2D)culturing conditions, prior to culturing of said adherent cells in said3D culture.
 32. The dispenser device of claim 22, wherein said adherentcells are less committed to an adipogenic lineage as compared toadherent cells from bone marrow grown and allowed to differentiate underthe same conditions.
 33. The dispenser device of claim 22, wherein saidadherent cells do not express CD34.
 34. The dispenser device of claim22, wherein the composition further comprises albumin.
 35. The dispenserdevice of claim 22, wherein the composition further comprises acryopreservent.